Outer membrane vesicles and uses thereof

ABSTRACT

The present invention discloses a Gram negative bacterium in which the expression of a protein involved in LPS transport to the outer membrane is functionally downregulated such that the level of LPS in the outer membrane is decreased compared to a wild-type Gram negative bacterium. Down regulation of Imp and MsbA proteins can result in such a bacterium. Outer membrane vesicle preparations derived from the Gram negative bacterium of the invention can be used in vaccines to provide protection against bacterial infection.

This Application is a §371 of International Application No.PCT/EP2004/014770, filed 21 Dec. 2004.

The present invention relates to Gram negative bacteria in which theexpression of a protein involved in the transport of lipopolysaccharide(LPS) to the outer membrane of the bacterium is functionallydownregulated. Examples of such proteins are the Imp and MsbA proteins.The invention also relates to Neisserial strains containing mutated impand/or msbA genes which exhibit disruption in lipopolysaccharide (LPS)transport to the outer membrane and/or contain a lower amount of LPS. Afurther aspect of the invention relates to outer membrane vesiclepreparations made from such strains. The present invention includesmutated Imp proteins and particularly chimeric Imp proteins. Theinvention also relates to vaccines and immunogenic compositionscontaining mutated Imp proteins or whole bacteria or fractions ofbacteria with disruption of transport of LPS to the outer membrane andtheir use in the treatment or prevention of Neisserial infection.

BACKGROUND

Gram negative bacteria are the causative agents for a number of humanpathologies and there is a need for effective vaccines to be developedagainst many of these bacteria. In particular Bordetella pertussis,Borrelia burgdorferi, Brucella melitensis, Brucella ovis, Chlamydiapsittaci, Chlamydia trachomatis, Esherichia coli, Haemophilusinfluenzae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseriameningitidis, Pseudomonas aeruginosa and Yersinia enterocolitica areGram negative bacteria which cause pathologies which could be treated byvaccination.

Neisseria meningitidis is an important pathogen, particularly inchildren and young adults. Septicemia and meningitis are the mostlife-threatening forms of invasive meningococcal disease (IMD). Thisdisease has become a worldwide health problem because of its highmorbidity and mortality.

Thirteen N. meningitidis serogroups have been identified based onantigenic differences in the capsular polysaccharides, the most commonbeing A, B and C which are responsible for 90% of disease worldwide.Serogroup B is the most common cause of meningococcal disease in Europe,USA and several countries in Latin America.

Vaccines based on the capsular polysaccharide of serogroups A, C, W andY have been developed and have been shown to control outbreaks ofmeningococcal disease (Peltola et al 1985 Pediatrics 76; 91-96). Howeverserogroup B is poorly immunogenic and induces only a transient antibodyresponse of a predominantly IgM isotype (Ala'Aldeen D and Cartwright K1996, J. Infect. 33; 153-157). There is therefore no broadly effectivevaccine currently available against the serogroup B meningococcus whichis responsible for the majority of disease in most temperate countries.This is particularly problematic since the incidence of serotype Bdisease is increasing in Europe, Australia and America, mostly inchildren under 5. The development of a vaccine against serogroup Bmeningococcus presents particular difficulties because thepolysaccharide capsule is poorly immunogenic owing to its immunologicsimilarity to human neural cell adhesion molecule. Strategies forvaccine production have therefore concentrated on the surface exposedstructures of the meningococcal outer membrane but have been hampered bythe marked variation in these antigens among strains.

Further developments have led to the introduction of vaccines made up ofouter membrane vesicles which will contain a number of proteins thatmake up the normal content of the bacterial membrane. One of these isthe VA-MENGOC-BC ® Cuban vaccine against N. meningitidis serogroups Band C (Rodriguez et al 1999 Mem Inst. Oswaldo Cruz, Rio de Janeiro 94;433-440). This vaccine was designed to combat an invasive meningococcaldisease outbreak in Cuba which had not been eliminated by a vaccinationprogramme using a capsular polysaccharide AC vaccine. The prevailingserogroups were B and C and the VA-MENGOC-BC ® vaccine was successful atcontrolling the outbreak with an estimated vaccine efficiency of 83%against serogroup B strains of N. meningitidis (Sierra et al 1990 InNeisseria, Walter Gruyter, Berlin, m. Atchman et al (eds) p 129-134,Sierra et al 1991, NIPH Ann 14; 195-210). This vaccine was effectiveagainst a specific outbreak, however the immune response elicited wouldnot protect against other strains of N. meningitidis.

Subsequent efficacy studies conducted in Latin America during epidemicscaused by homologous and heterologous serogroup B meningococcal strainshave shown some efficacy in older children and adults but itseffectiveness was significantly lower in younger children who are atgreatest risk of infection (Milagres et al 1994, Infect. Immun. 62;4419-4424). It is questionable how effective such a vaccine would be incountries with multistrain endemic disease such as the UK. Studies ofimmunogenicity against heterologous strains have demonstrated onlylimited cross-reactive serum bactericidal activity, especially ininfants (Tappero et al 1999, JAMA 281; 1520-1527).

A second outer membrane vesicle vaccine was developed in Norway using aserotype B isolate typical of those prevalent in Scandinavia (Fredriksenet al 1991, NIPH Ann, 14; 67-80). This vaccine was tested in clinicaltrials and found to have a protective efficacy after 29 months of 57%(Bjune et al 1991, Lancet, 338; 1093-1096).

However, the use of outer membrane vesicles in vaccines is associatedwith some problems. For instance, the OMV contain toxiclipopolysaccharides (LPS). The toxicity of outer membrane vesicles maybe decreased by treatment with detergents to remove the majority of LPSin order to prevent toxic reactions in vaccinees. This procedureunfortunately also removes other potentially important vaccinecomponents such as surface exposed lipoproteins.

The imp gene encodes the Imp/OstA protein which is an outer membraneprotein of Gram negative bacteria. Imp/OstA has been most extensivelystudied in E. coli where it was first described as having a role inouter membrane permeability (Sampson et al 1989 Genetics 122, 491-501).Imp/OstA was subsequently found to determine organic solvent tolerancein E. coli (Aono et al 1994 Appl. Environ. Microbiol. 60, 4624-4626). Ithas been proposed that Imp/OstA contributes to n-hexane resistance of E.coli by reducing the influx of n-hexane (Abe et al 2003, Microbiology149, 1265-1273).

The msbA gene was first identified in E. coli as a multicopy-suppressorof the mutation in the htrB (IpxL) gene, which encodes an enzymeinvolved in a late step of lipid A biosynthesis (Karow andGeorgeopoulos, 1993. Mol Microbiol. 7, 69-79). The MsbA protein belongsto a family of ABC (ATP-binding cassette) transporters. Atemperature-sensitive msbA mutant of E. coli has been reported toaccumulate LPS as well as three major PL in the inner membrane whenshifted to the restrictive growth temperature (Doerrier, et al 2001 J.Biol. Chem. 276, 11461-11464). This result indicated a role for MsbA inthe translocation of both LPS and PL across the inner membrane and/or,as proposed earlier (Polissi and Georgopoulos, 1996 Mol. Microbiol. 20,1221-1233), in a later step of the transport process.

There is a need for improved vaccines for use in treatment andprevention of Gram negative bacterial infection, particularly Neisserialinfection. It is particularly important to address the problem of LPStoxicity in vaccines comprising whole bacteria, or outer membranevesicle preparations whilst ensuring that desirable antigens areretained in the outer membrane. The present application discloses thegeneral concept of outer membrane vesicle vaccines prepared from Gramnegative bacterial mutant strains, particularly Neisserial strains suchas N. meningitidis, which have reduced LPS compared to wild typestrains, or no LPS on its surface. Such vaccines have the advantage thatthe outer membrane vesicles may be produced using a protocol involvingextraction with low or no detergent thus retaining protective antigenssuch as lipoproteins on the outer membrane vesicle surface. It isparticularly preferred if a low level (less than 50, 40, 30, 20 or 10%of wild-type level) of LPS is maintained in the mutant strain so thatone or both of the following advantages are realised: i) the LPS canstill be used as an antigen in its own right, and ii) the strain maygrow better for production purposes. The inventors have found thatdisruption of either the Imp or MsbA proteins can produce such strainsand outer membrane vesicle vaccines. A particularly preferred mutant forthese purposes is a functional disruption of the imp gene.

The present invention further provides a mutated Imp or MsbA protein,for example a chimeric protein comprising a backbone polypeptide whichis derived from an Imp protein and at least one insert region derivedfrom a different protein wherein part or all of at least one Impextracellular loop is replaced with one or more polypeptide sequencefrom at least one additional protein. Also provided are vaccinecomponents comprising a chimera of part or all of at least one Impextracellular loop with a different carrier protein which providesT-helper epitopes.

The present application discloses proteins that regulate the transportof LPS to the outer membrane of Gram negative bacteria. In particular, afunction has been provided for Imp in regulating the transport of LPS tothe outer membrane of Gram negative bacteria. It further discloses thatMsbA regulates the transport of LPS to the outer membrane of Neisseriaand the disruption of this protein does not lead to a disruption ofphospholipid transport to the outer membrane. Downregulation of Imp orMsbA, either by downregulation of expression of the imp or msbA gene orby disrupting the structure of the Imp or MsbA protein so that it nolonger transports LPS to the outer membrane, leads to most (but not all)of the LPS failing to reach the cell surface as shown in FIGS. 5 and 10.Downregulation of Imp MsbA also leads to a decrease in the amount of LPSpresent in the bacteria due to feedback inhibition on LPS synthesis bymislocalised LPS. Downregulation of Imp or MsbA therefore produces aGram negative bacterium (preferably a Neisserial bacterium) with a lowlevel of LPS, equivalent or lower to the level achieved after detergenttreatment. Such a bacterium has lower toxicity whilst retainingsufficient LPS to enable the LPS to contribute to the immunogenicity ofthe bacterium/vaccine composition.

A further advantageous aspect of some embodiments of the invention isthat the Imp protein is used as a scaffold to display advantageousheterologous antigens on the outer membrane of Gram negative bacteria,preferably a Neisserial strain, more preferably N. meningitidis. Theseantigens are positioned at the site of one of the Imp extracellular(surface exposed) loops.

A further advantage of some embodiments of the invention is realisedwhen at least some of the extracellular loops of Imp are retained in thechimeric protein of the invention. The amino acid sequence of theextracellular loops are well conserved and antibodies against anextracellular loop of Imp should crossreact with a wide range ofbacterial, preferably Neisserial strains.

In a preferred embodiment, the invention provides a Gram negativebacterium in which a protein involved in the transport of LPS to theouter membrane, for instance Imp or MsbA, is down regulated such thatLPS transport to the outer membrane is disrupted.

In a further embodiment, the invention provides a polynucleotidecomprising a sequence encoding the mutated or chimeric protein of theinvention, an expression vector comprising a sequence encoding thechimeric protein of the invention and a host cell comprising saidexpression vector. Polynucleotides of the invention do not encompass abacterial genome.

In a further embodiment, the invention provides an outer membranevesicle preparation, from a strain in which the expression of a proteinregulating LPS transport to the outer membrane, for instance Imp orMsbA, is downregulated such that the outer membrane vesicle has a lowerLPS content than outer membrane vesicles derived from a similar strainof Gram negative bacterium in which transport of LPS to the outermembrane has not been disrupted.

In a further embodiment, the invention provides a method for producingthe chimeric protein or outer membrane vesicle preparation of theinvention.

In a further embodiment, the invention provides a pharmaceuticalpreparation, preferably a vaccine comprising the Gram negative(preferably Neisserial) bacterium of the invention or a fraction ormembrane thereof, the chimeric protein of the invention, or the outermembrane vesicle preparation of the invention, and a pharmaceuticallyacceptable carrier.

In a further embodiment, the invention provides methods of treatment orprevention of Gram negative bacterial infection, preferably Neisserialinfection.

DESCRIPTION OF DRAWINGS

FIG. 1. Construction of an imp mutant strain. (A) Genomic organizationof the imp locus in the wild-type (WT) and imp mutant. NMB0279 isannotated as a conserved hypothetical protein in the MC58 database(http://www.tigr.org). The surA gene (survival protein A) encodes aperiplasmic chaperone involved in OMP biogenesis. rnb: ribonuclease II.Arrows indicate the DNA region used for transformation. (B) Immunoblotof cell envelopes of wild-type (lane 1) and imp mutant (lane 2)separated on 8% SDS-PAGE and probed with anti-Imp antibodies. Molecularsize markers are indicated in kDa.

FIG. 2. Characteristics of an Nme imp mutant. (A-C) Colony morphology ofwild-type (A), imp mutant (B) and IpxA mutant (C) bacteria. Colonieswere observed with a binocular microscope using the shiny side of aflexible mirror. (D) Growth curve of wild-type (¦) and imp mutant (.)bacteria in TSB.

FIG. 3. Protein and LPS profiles of wild-type (lanes 1), imp mutant(lanes 2) and IpxA mutant (lanes 3) bacteria. (A, B) Cell envelopes wereanalysed by 10% SDS-PAGE in denaturing (95° C.+) or semi-native (95°C.−) conditions. Gels were stained with Coomassie blue (A) or blottedand probed with anti-PorA antibody (B). (C) Equal amounts of proteinaseK-treated whole cell lysates were subjected to Tricine-SDS-PAGE andstained with silver to visualize LPS. (D) Equal volumes of extracellulargrowth media (100.000 g supernatant) were precipitated with TCA,subjected to 11% SDS-PAGE and stained with Coomassie blue. Molecularsize markers (in kDa) are indicated.

FIG. 4. Analysis of fractions obtained after isopycnic sucrose gradientcentrifugation of wild-type Nme membranes. (A) Percentage sucrose (.),measured in a refractometer and LDH activity (¦) in the differentfractions. (B, C) Equal volumes of each fraction were precipitated withTCA and separated in denaturing SDS-PAGE followed by Coomassie bluestaining (B) or separated on Tricine-SDS-PAGE followed by silverstaining to visualize LPS (C). The positions of the major OMPs PorA andPorB are indicated. Molecular size markers are indicated in kDa.

FIG. 5. Surface accessibility of LPS. All panels show silver-stainedTricine SDS-PAGE gels containing samples treated with proteinase Kbefore loading. (A) Equal amounts of whole cell lysates of the indicatedstrains were loaded on the same gel. (B) Cell envelopes of bacteriagrown the presence or absence of 80 μM CMP-NANA. Where indicated, thecell envelopes were treated with neuraminidase before electrophoresis.(C) Intact bacteria grown in the presence of 80 μM CMP-NANA were treatedwith neuraminidase and subsequently processed for Tricine-SDS-PAGE. Inpanels B and C five times as much material of the imp mutant samples wasloaded compared to wild-type samples. Wild type and imp mutant sampleswere electrophoresed and stained on separate gels, to obtain optimalvisibility of the LPS bands of both variants. (D) The inducible IpxAmutant was grown in the presence of the indicated IPTG concentrationsplus 80 μM CMP-NANA. Intact cells were treated with neuraminidase asindicated. Equal amounts of cell lysates were run on the same gel.

FIG. 6. Topology models of Neisserial Imp.

FIG. 7. Sequence of Imp (SEQ ID NO. 1) showing position of the nineextracellular loops.

FIG. 8. Alignment of meningococcal Imp sequences.

FIG. 9. Genetic organization of the msbA locus in the wild-type strainand the constructed msbA mutant.

The kanamycin-resistance cassette (KAN) replaces msbA in the mutant,leaving only 131 bp at the 3′ end (M). Primers used for the disruptionprocedure and cloning of msbA are indicated with arrows. Primersequences are (A) CCCAAAGCGAAGTGGTCGAA (SEQ ID NO: 6); (B)GTCGACTATCGGTAGGGCGGGAACTG (AccI restriction site is underlined)(SEQ IDNO: 7); (C) GTCGACGACCGCATCATCGTGATGGA (AccI restriction site isunderlined)(SEQ ID NO: 8)(D); TTCGTCGCTGCCGACCTGTT (SEQ ID NO: 9); (E)TTCATATGATAGAAAAACTGACTTTCGG (NdeI restriction site is underlined) SEQID NO: 10); (F) GACGTCCCATTTCGGACGGCATTTTGT (AatII restriction site isunderlined) (SEQ ID NO: 11). Predicted promoter (P) and terminator (T)sequences are indicated. ORFs indicated with NMB1918 and NMB1920putatively code for a malonyl CoA-acyl carrier protein transacylase andGMP synthase, respectively.

FIG. 10. LPS content in the msbA mutant.

A. Cells from strain HB-1 (WT) and its msbA-mutant derivative (ΔmsbA)were resuspended from plate and the LPS content was analyzed byTricine-SDS-PAGE.

B. KDO and protein concentrations were measured from cell envelopesisolated from different strains derived from H44/76. The KDOconcentrations measured were corrected for the background value measuredin the IpxA mutant, and the ratio of the LPS and protein concentrationin the wild-type strain was set to 100%.

FIG. 11. Growth of the msbA mutant.

Strain HB-1 (wild type) and its msbA -mutant derivative were grown onplate overnight and resuspended in 5 ml of TSB. The OD₅₅₀ was measuredin time during incubation at 37° C. while shaking at 180 rpm.

FIG. 12. Morphology and cell envelope protein profile of the msbAmutant.

A. Electron micrograph of an ultrathin section of the msbA mutantderived from H44/76. The area inside the white rectangle is shown at ahigher magnification in panel B. The inner (IM) and outer membrane (OM)are indicated with arrows. Scale bars are 100 nm. C. Cell envelopeprotein profiles of wild-type strain H44/76 (lane 1), its msbA mutantderivative (lane 2) and the msbA mutant complemented with pEN 11 -msbA(lane 3). PorA and PorB are indicated at the left.

FIG. 13. Phospholipids analysis of wild-type and msbA-mutant strain.

A. Cells from strain HB-1 (WT) and its msbA-mutant derivative (.msbA)were labeled with [14C] acetate, and their phospholipids were isolatedand analyzed by TLC. The positions of the major PL species areindicated.

B. Cells grown on plate were resuspended, and, based upon the OD550,equal amounts of cells were used for PL isolation. The PL werequantified for their phosphorus content. Wild-type amounts were set at100% and compared with amounts isolated from the msbA mutant. Meanvalues are derived from 6 independent experiments.

FIG. 14. A—Amino Acid sequence of MsbA from N. meningitidis (SEQ IDNO:2).

-   -   B—Amino Acid sequence of MsbA from B. parapertussis (SEQ ID        NO:4).

FIG. 15. A—Nucleic acid sequence of MsbA from N. meningitidis (SEQ IDNO:3).

-   -   B—Nucleic acid sequence of MsbA from B. pertussis (SEQ ID NO:5).

DETAILED DESCRIPTION

The terms “comprising”, “comprise” and “comprises” herein are intendedby the inventors to be optionally substitutable with the terms“consisting of”, “consist of” and “consists of”, respectively, in everyinstance.

The terms lipopolysaccharide (LPS) and lipooligosaccharide (LOS) areinterchangeable and the correct term for the bacterial strain inquestion should be adopted.

Gram Negative Bacterium with Reduced LPS Transport to the Outer Membrane

One aspect of the invention is a Gram negative bacterium in which theexpression of a protein involved in LPS transport to the outer membraneis downregulated such that the level of LPS in the outer membrane isdecreased compared to a wild-type Gram negative bacterium or such thatLPS transport to the outer membrane is disrupted. Examples of proteinsinvolved in LPS transport to the outer membrane are Imp and MsbA. 1, 2,3, 4 or 5 or more proteins involved in LPS transport to the outermembrane may be functionally downregulated.

The wild-type Gram negative bacterium is defined as the correspondingGram negative bacterium in which the expression of proteins involved inLPS transport to the outer membrane has not been disrupted.

Functional downregulation of the protein involved in LPS transportshould not result in a lethal phenotype. For instance, in the case ofMsbA downregulation, the Gram negative bacterium is preferably not astrain of E. Coli in which phospholipids transport is disrupted.

Imp and/or MsbA expression is downregulated by either downregulatingexpression from the imp and/or msbA gene or by disrupting the structureof the Imp and/or MsbA protein so that it no longer transports LPS tothe outer membrane efficiently, i.e. so that the amount of LPS presentin the outer membrane is reduced.

Downregulated preferably means functionally downregulated. This may beaccomplished by downregulation of expression, or disruption of the geneso that no expression occurs. It may also be accomplished by alteringthe structure of the protein involved in transport of LPS to the outermembrane (e.g. Imp or MsbA) by deletion of amino acids, insertion ofamino acids or substitution of amino acids so that the resultant proteintransports LPS to the outer membrane of a Gram negative bacterium lesseffectively that the unmutated protein.

The functional downregulation of the protein involved in the transportof LPS to the outer membrane (e.g. Imp or MsbA) results in a decrease ofthe amount of LPS on the outer membrane of at least 10%, 20%, 30%, 40%,50%, 60%, 70%, preferably 80%, 90% or 95% or 99% or 100% compared to asimilar strain of Gram negative bacteria in which Imp and/or MsbA is notdown regulated.

Where the level of expression of the protein involved in the transportof LPS to the outer membrane (e.g. Imp or MsbA) is disrupted, the amountof the protein involved in the transport of LPS to the outer membrane(e.g. Imp or MsbA) in the outer membrane is decreased by at least 20%,30%, 40%, 50%, preferably 60%, 70%, 80%, 90%, 95%, 98% or 100%.Optionally the level of expression of both Imp and MsbA is disrupted.

In a preferred embodiment, the Gram negative bacterium of the inventioncomprises a mutated protein involved in the transport of LPS to theouter membrane (for instance, Imp and/or MsbA) in which the structure ofthe protein is disrupted by removing part of the sequence to form atruncated protein, or by changing the sequence so that LPS transportingactivity is decreased or lost, or by deleting part of the sequence ofthe protein and replacing it with a sequence from a different protein tomake a chimeric protein.

In a preferred embodiment, at least part of at least 1, 2, 3, 4, 5, 6,7, 8 or 9 extracellular loops of the Imp protein are removed. Thedeleted sequence(s) are optionally replaced with the sequence from adifferent protein to make a chimeric protein. The inventors have foundthat Imp protein provides a very good scaffold for the display ofheterologous peptide or epitopes in a useful conformation, particularlywhen inserted into or if replacing an Imp extracellular loop. Such Impproteins and outer membrane vesicles containing these, form anindependent aspect of the present invention.

The Gram negative bacterium of the invention preferably comprises atleast one of the mutated or chimeric proteins of the invention describedbelow.

The Gram negative bacterium is selected from any suitable strain of Gramnegative bacterium. Where Imp expression is targeted, the wild-type Gramnegative bacterium must express an Imp homolog (therefore for thisaspect of the invention Gram negative bacteria are not from Thermotogamaritima, Deinococcus radiodurans, Borrelia burgdorfferi or Treponemapalladium). Where MsbA expression is targeted, the MsbA downregulationdoes not lead to a lethal phenotype, therefore for this aspect of theinvention, the Gram negative bacterium is not Esherichia coli. PreferredGram negative bacteria include Bordetella pertussis, Moraxellacatarrhalis, Brucella melitensis, Brucella ovis, Chlamydia psittaci,Chlamydia trachomatis, Esherichia coli, Haemophilus influenzae,Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis,Pseudomonas aeruginosa and Yersinia enterocolitica. Most preferably, theGram negative bacterium is Neisseria meningitidis.

Proteins and Chimeric Proteins

A further aspect of the invention is a mutated MsbA or Imp protein inwhich the function of transporting LPS to the outer membrane has beendisrupted. This may be achieved by deleting regions of MsbA and/or Impto form truncated proteins or by mutating amino acids within thepolypeptide sequence. A chimeric protein wherein one or more region(s)of the sequence of MsbA or Imp are exchanged for sequence from otherprotein(s) may also be used to disrupt the function of transporting LPSto the outer membrane.

A chimeric protein is a protein containing polypeptide sequence derivedfrom two or more different proteins. It contains a backbone polypeptideinto which sequence derived from at least one other protein is insertedor adjoined. The backbone polypeptide typically makes up the majority ofthe chimeric protein and in the case of the present invention, isderived from a protein involved in the transport of LPS to the outermembrane, for instance an Imp or an MsbA protein. In some embodiments ofthe invention, the protein involved in LPS transport to the outermembrane (e.g. Imp or MsbA) will make up a small fraction of thechimeric protein.

Where the chimeric protein of the invention is an Imp mutant, itcomprises at least one part (optionally at least 2, 3, 4, 5, 6, 7, 8, 9or 10 parts) which are derived from an Imp protein and at least one part(optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 parts) which arederived from at least one different protein (optionally at least 2, 3,4, 5, 6, 7, 8, 9 or 10 different proteins).

‘Derived from’ indicates the origin of the protein sequence. A derivedsequence encompassed both the complete protein sequence and a portion ofthe complete protein sequence.

Preferred embodiments of the invention include chimeric proteins inwhich the majority of the protein is Imp and the extracellular loops areadapted to carry peptides from other proteins, optionally by deleting atleast some of the Imp extracellular loop(s). They also include chimericproteins containing at least one extracellular loop(s) of Impincorporated into the structure of a different protein, preferably abacterial outer membrane protein. They also include at least oneextracellular loop from Imp linked to a carrier of T-cell epitopes. Thelink could be a peptide bond, covalent bonds formed by a conjugationprocess, preferably as described below, or non-covalent interactions.

Optionally, the chimeric protein is derivable from the Gram negativebacterium of the invention.

Preferably, the Gram negative bacteria and chimeric proteins of theinvention contain an Imp or MsbA polypeptide derived from any Gramnegative bacterium, preferably from Bordetella pertussis, Moraxellacatarrhalis, Brucella melitensis, Brucella ovis, Chlamydia psittaci,Chlamydia trachomatis, Esherichia coli, Haemophilus influenzae,Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis,Pseudomonas aeruginosa and Yersinia enterocolitica, most preferably fromNeisseria meningitidis. Most preferably, a chimeric protein of theinvention contains an Imp polypeptide with a sequence sharing at least70%, 80%, 85%, 90%, 95% or 99% identity with the corresponding sequenceof SEQ ID NO. 1. Most preferably, a chimeric protein of the inventioncontains an MsbA polypeptide with a sequence sharing at least 70%, 80%,85%, 90%, 95% or 99% identity with the corresponding sequence of SEQ IDNO. 2. Since the chimeric protein contains only part of the sequence ofthe Imp or MsbA protein, the degree of sequence identity is calculatedon the basis of corresponding sequences. This means that the parts ofthe Imp or MsbA sequence deleted and/or replaced are not included inthis sequence identity calculation.

Alternatively, where the Imp or MsbA polypeptide makes up at least 50%of the chimeric protein, the complete sequence of the chimeric proteinshares at least 40%, 50%, 60%, preferably 70%, 75%, 80%, 85%, 90% or 95%with the sequence of SEQ ID NO: 1 or SEQ ID NO:2.

The inventors have elucidated a topology model of Imp which indicatesthe presence of 9 extracellular (surface-exposed) loops. At least someamino acids from at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the surfaceloops of the Imp protein may be replaced by non-natural, i.e.heterologous sequence as an insert. At least some of any of the loopsmay be replaced with heterologous sequence, however, preferred loops tobe inserted into, replaced, altered or deleted are one or more of loop3, loop 8, loop 6 and loop 2. Preferred combination of loops to changeinclude loop 3; loop 8; loops 3 and 8; loop 6; loops 3 and 6; loops 6and 8; loops 3, 6 and 8; loop 2; loops 2 and 3; loops 2 and 6; loops 2and 8; loops 2, 3 and 6; loops 2, 3 and 8; loops 2, 6 and 8; loops 2, 3,6 and 8. The preferred combinations of loops replaced by heterologoussequence (or altered or deleted) are optionally combined withreplacement (or alteration of deletion) of one or more of loops 1, 4, 5,7 and 9. In a further preferred embodiment at least some of all 9 loopsare deleted or deleted and replaced with heterologous sequence.

The size of deletion of the extracellular loop is at least 6, 10, 15,20, 30, 40 or 50 amino acids. The deleted sequence is optionallyreplaced with an insert sequence of at least 6, 10, 15, 20, 30, 40, 50,60 or 70 amino acids.

Preferred chimeric proteins contain an Imp backbone in which sequence(s)corresponding to 1, 2, 3, 4, 5, 6, 7, 8 or 9 of; amino acids 357-416,648-697, 537-576, 295-332, 252-271, 444-455, 606-624, 482-501,or 721-740of SEQ ID No 1 is/are absent from the backbone polypeptide. At least 6,10, 15, 20, 30, 40 or 50 amino acids may be absent from one or more ofthe above sequences.

The replacement sequence or insert (if employed) is from a differentprotein. It can be from the same strain or a different strain ofbacteria and is preferably from a bacterial outer membrane protein. Itis preferred that such replacement sequences are conserved and/orsurface exposed, i.e. able to generate an immune response, preferablyagainst more than one strain of a bacterial organism. Preferably, oneloop or part thereof, is replaced with an insert sequence from a singleprotein. Where multiple loops are replaced, they are preferably replacedwith inserts from different proteins or the same protein from differentstrains of bacteria, preferably Neisseria.

In one embodiment the replacement sequence is derived from Neisserialouter membrane proteins, such as Neisseria gonorrhoeae or Neisseriameningitidis. An example of such a suitable outer membrane protein isgiven in U.S. Pat. No. 5,912,336 which describes a Neisserial ironregulated protein, designated TbpA. Replacement sequence couldconveniently be derived from any one or more of loops 2, 3, 4, 5 and 8of TbpA. These loops correspond generally to amino acids 226-309;348-395; 438-471; 512-576 and 707-723 of TbpA respectively. Preferablyone or more of loops 4, 5 and 8 are incorporated. An insert is derivedfrom TbpA-high molecular weight and/or TbpA-low molecular weight (asdescribed later). In a preferred embodiment, an insert of TbpA-highmolecular weight replaces at least part of an Imp extracellular loop andan insert of TbpA-low molecular weight replaces at least part of adifferent Imp extracellular loop. Preferably the preferred loopcombinations described above are replaced.

Another example of such a suitable outer membrane protein is given inWO01/55182, which describes the NhhA (or Hsf) surface antigen fromNeisseria meningitidis. Replacement sequence could conveniently bederived from one or more constant regions of an NhhA protein generallydesignated as C1, C2, C3, C4 and C5. An example of another replacementsequence which could be used in the present invention is described in EP0 586 266.

Further Neisserial OMP loops that may be substituted for Imp loops(particularly loops 3 and/or 8) are PorA loop 4 [or variable region 2] ;PorA loop 5 (described in “Topology of outer membrane porins inpathogenic Neisseria spp”, van der Ley, Poolman, etc., Infect Immun1991, 59, 2963-71; its sequence in PorA P1.7,16 (H44/76) loop 5 being:RHANVGRNAFELFLIGSGSDQAKGTDPLKNH, SEQ ID NO: 12); LbpA surface exposedloops 4, 5, 7, 10 and 12, corresponding to amino acids 210-342, 366-441,542-600, 726-766 and 844-871, respectively, with 12 being preferred(sequence KGKNPDELAYLAGDQKRYSTKRASSSWST), SEQ ID NO: 13) [see Prinz etal. 1999 J Bacter. 181:4417 for further details on LbpA surface loopsincorporated by reference herein]; NspA surface exposed loops 1, 2, 3 or4, corresponding to amino acid sequence 25-54, 61-87, 103-129 and149-164, respectively, preferably where loop 2 (e.g.FAVDYTRYKNYKAPSTDFKLYSIGASA, SEQ ID NO: 14) and/or 3 (e.g.ARLSLNRASVDLGGSDSFSQTSIGLGVL, SEQ ID NO: 15) is inserted (as these loopsare quite small not all the Imp loop 2 and/or 8 would be ideally removedto introduce these loops, and if both are to be introduced, it ispreferred that they are introduced on loop 2 or 8 (or vice versa) inorder to try to preserve the conformational epitope that exists betweenloops 2 and 3 of NspA) [see Vandeputte-Rutten et al 2003 JBC 278:24825for more details on NspA loops, incorporated by reference herein]; anyof the surface exposed loops of Omp85 (see Science 2003 299:262-5, andsupporting online material FIG. S4, incorporated by reference herein).

Alternatively peptide mimotopes of bacterial carbohydrate antigens maybe incorporated into Imp in the above way. Preferably mimotopes ofNeisserial LOS are incorporated into loops 2 and/or 8 to advantageouslystimulate an immune response against this important antigen withouthaving its toxic effects in a vaccine. LOS mimotopes are well known inthe art (see WO 02/28888 and references cited therein, incorporated byreference herein).

In a preferred embodiment of the invention, the chimeric proteincomprises all or part of at least one extracellular loop from Imp. Asshown in FIG. 7, the Imp protein is well conserved between Gram negativebacterial strains and is therefore an antigen that elicitscross-reactive antibodies which react with different strains of Gramnegative bacteria, preferably Neisseria. Preferably the chimeric proteinof the invention comprises at least 6, 10, 15, 20, 30, 40 or 50 aminoacids of at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the Imp extracellularloops 1, 2, 3, 4, 5, 6, 7, 8 and/or 9. Preferred combinations of Impextracellular loops to be retained are loops 3 and 8, loops 3 and 6,loops 6 and 8, loops 3, 6 and 8 or all 9 extracellular loops.

In one embodiment of the invention, the extracellular loop(s) of Imp(preferably substantially devoid of Imp sequence not part of anextracellular loop) is covalently linked to sequence from a differentprotein. This may be achieved through peptide bonds linking thepolypeptide sequence of at least one Imp extracellular loop to thepolypeptide sequence of at least one different protein (acting as acarrier) to form a chimeric protein. Alternatively, the Impextracellular loop(s) is conjugated to a carrier molecule, preferably aprotein or a polysaccharide or oligosaccharide or lipopolysaccharideusing conjugation methods as described below. The carrier is preferablya protein comprising T-cell epitopes, such as tetanus toxoid, tetanustoxoid fragment C, diphtheria toxoid, CRM197, pneumolysin, Protein D(U.S. Pat. No. 6,342,224).

It will be appreciated that the mutant proteins of the present inventionmay be prepared using conventional protein engineering techniques. Forexample, polynucleotides of the invention or coding for a wild-type Impmay be mutated using either random mutagenesis, for example usingtransposon mutagenesis, or site-directed mutagenesis.

It will be understood that protein sequences of the invention or for usein the invention are provided as guidelines and the invention is notlimited to the particular sequences or fragments thereof given here butalso include homologous sequences obtained from any source, for examplerelated bacterial proteins, and synthetic peptides, as well as variants(particularly natural variants) or derivatives thereof. Loop sequencesgiven are meant as guidelines, and it is envisaged that any loopsequence comprising an epitope present in the loops described above maybe utilised.

Thus, the present invention encompasses variants, homologues orderivatives of the amino acid sequences of the present invention or foruse in the invention, as well as variants, homologues or derivatives ofthe amino acid sequences.

In the context of the present invention, a homologous sequence is takento include an amino acid sequence which is at least 60, 70, 80 or 90%identical, preferably at least 95 or 98% identical at the amino acidlevel. Although homology can also be considered in terms of similarity(i.e. amino acid residues having similar chemical properties/functions),in the context of the present invention it is preferred to expresshomology in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues (for example less than 50 contiguousamino acids).

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage (see below) the default gap penalty for amino acid sequences is−12 for a gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However it is preferred to use the GCG Bestfit program.

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). It is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

Where a protein is specifically mentioned herein, it is preferably areference to a full-length protein but it may also encompass antigenicfragments thereof (particularly in the context of subunit vaccines).Preferred fragments include those which include an epitope. Particularlypreferred fragments include those with at least one surface loop. Withrespect to the mutants of the present invention this loop is preferablyother than loop 7 and/or loop 5. These fragments may contain or compriseat least 10 amino acids, preferably 20 amino acids, more preferably 30amino acids, more preferably 40 amino acids or most preferably 50 aminoacids, taken contiguously from the amino acid sequence of the protein.In addition, antigenic fragments denotes fragments that areimmunologically reactive with antibodies generated against theNeisserial proteins (or other Gram negative bacteria) or with antibodiesgenerated by infection of a mammalian host with Neisseria. Antigenicfragments also includes fragments that when administered at an effectivedose, elicit a protective immune response against Neisserial (or otherGram negative bacterial) infection, more preferably it is protectiveagainst N. meningitidis and/or N. gonorrhoeae infection, most preferablyit is protective against N. meningitidis serogroup B infection.

The present invention also includes variants of the proteins mentionedherein, that is proteins that vary from the referents by conservativeamino acid substitutions, whereby a residue is substituted by anotherwith like characteristics. Typical such substitutions are among Ala,Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp andGlu; among Asn and Gln; and among the basic residues Lys and Arg; oraromatic residues Phe and Tyr. Particularly preferred are variants inwhich several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted,deleted, or added in any combination.

The chimeric protein produced by the present invention is preferably aproduct which displays at least some of the immunological activity ofthe wild type Imp protein. Preferably it will show at least one of thefollowing:

-   An ability to induce the production of antibodies which recognise    the wild type Imp (if necessary when the Imp protein of the present    invention is coupled to a carrier);-   An ability to induce the production of antibodies that can protect    against experimental infection; and/or-   An ability to induce, when administered to an animal, the    development of an immunological response that can protect against    Gram negative bacterial infection, preferably Neisserial infection    such as Neisseria meningitidis or Neisseria gonorrhoeae infection.

Preferably the mutant protein of the present invention is cross-reactiveand more preferably cross-protective.

The chimeric protein of the present invention is useful in prophylactic,therapeutic and diagnostic composition for preventing treating anddiagnosing diseases caused by Gram negative bacteria, preferablyNeisseria, particularly Neisseria meningitidis; although it may alsohave similar applications in relation to, e.g. Neisseria gonorrhoeae orNeisseria lactamica.

Standard immunological techniques may be employed with the chimericprotein of the present invention in order to use it as an immunogen andas a vaccine. In particular, any suitable host may be injected with apharmaceutically effective amount of the chimeric protein to generatemonoclonal or polyclonal anti-Imp antibodies or to induce thedevelopment of a protective immunological response against a Neisseriadisease. Prior to administration, the chimeric protein may be formulatedin a suitable vehicle, and thus we provide a pharmaceutical compositioncomprising a pharmaceutically effective amount of one or more proteinsof the present invention. As used herein “pharmaceutically effectiveamount” refers to an amount of Imp (or other proteins of the invention)protein that elicits a sufficient titre of antibodies to treat orprevent infection. The pharmaceutical composition of the presentinvention may also comprise other antigens useful in treating orpreventing disease.

Polynucleotide

The present invention also provides polynucleotides which code for thechimeric proteins of the present invention, including variants,derivatives and homologs thereof. Polynucleotides of the invention maycomprise DNA or RNA. They may be single-stranded or double-stranded.They may also be polynucleotides which include within them synthetic ormodified nucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes of thepresent invention, it is to be understood that the polynucleotidesdescribed herein may be modified by any method available in the art.Such modifications may be carried out in order to enhance the in vivoactivity or life span of polynucleotides of the invention.

In one embodiment the mutant proteins of the present invention areproduced using any one of the following techniques: site-directedmutagenesis including cassette mutagenesis, single primer extension, aPCR method of site-directed mutagenesis for example the four-primermethod of Higuchi et al (1988) Nucleic Acids Res. 16:7351-67,unidirectional deletion; random mutagenesis; and selection of mutantproteins by phage display.

The terms “variant”, “homologue” or “derivative” in relation to thenucleotide sequence of the present invention include any substitutionof, variation of, modification of, replacement of, deletion of oraddition of one (or more) nucleic acid from or to the sequence providingthe resultant nucleotide sequence codes for a mutant Imp or MsbApolypeptide.

As indicated above, with respect to sequence homology, preferably thereis at least 75%, more preferably at least 85%, more preferably at least90% homology (preferably identity) to the polynucleotide sequences shownherein or there is at least 75%, more preferably at least 85%, morepreferably at least 90% homology (preferably identity) topolynucleotides encoding polypeptide sequences shown herein. Morepreferably there is at least 95%, more preferably at least 98%, homology(preferably identity). Nucleotide homology comparisons may be conductedas described above. A preferred sequence comparison program is the GCGWisconsin Bestfit program described above. The default scoring matrixhas a match value of 10 for each identical nucleotide and −9 for eachmismatch. The default gap creation penalty is −50 and the default gapextension penalty is −3 for each nucleotide.

The present invention also encompasses nucleotide sequences that arecapable of hybridising selectively to the sequences presented herein orto polynucleotides encoding the polypeptide sequences presented herein,or any variant, fragment or derivative thereof, or to the complement ofany of the above. Nucleotide sequences are preferably at least 15nucleotides in length, more preferably at least 20, 30, 40 or 50nucleotides in length.

The term “hybridization” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction technologies.

Polynucleotides of the invention capable of selectively hybridising tothe nucleotide sequences presented herein, polynucleotides encodingpolypeptide sequences presented herein, or to their complement, will begenerally at least 70%, preferably at least 80 or 90% and morepreferably at least 95% or 98% homologous to the correspondingnucleotide sequences presented herein over a region of at least 20,preferably at least 25 or 30, for instance at least 40, 60 or 100 ormore contiguous nucleotides. Preferred polynucleotides of the inventionwill comprise regions homologous to nucleotides which code for conservedregions, preferably at least 80 or 90% and more preferably at least 95%homologous (preferably identical) to these regions.

The term “selectively hybridizable” means that the polynucleotide usedas a probe is used under conditions where a target polynucleotide of theinvention is found to hybridize to the probe at a level significantlyabove background. The background hybridization may occur because ofother polynucleotides present, for example, in the cDNA or genomic DNAlibrary being screening. In this event, background implies a level ofsignal generated by interaction between the probe and a non-specific DNAmember of the library which is less than 10 fold, preferably less than100 fold as intense as the specific interaction observed with the targetDNA. The intensity of interaction may be measured, for example, byradiolabelling the probe, e.g. with ³²P.

Hybridization conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego, Calif.), and confer a defined “stringency” asexplained below.

Maximum stringency typically occurs at about Tm-5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridization can beused to identify or detect identical polynucleotide sequences while anintermediate (or low) stringency hybridization can be used to identifyor detect similar or related polynucleotide sequences.

In a preferred aspect, the present invention covers nucleotide sequencesthat can hybridise to the nucleotide sequence of the present inventionunder stringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl,0.015 M Na₃ Citrate pH 7.0}).

Where the polynucleotide of the invention is double-stranded, bothstrands of the duplex, either individually or in combination, areencompassed by the present invention. Where the polynucleotide issingle-stranded, it is to be understood that the complementary sequenceof that polynucleotide is also included within the scope of the presentinvention.

Polynucleotides which are not 100% homologous to the sequences of thepresent invention but fall within the scope of the invention can beobtained in a number of ways. Other variants of the sequences describedherein may be obtained for example by probing DNA libraries made from arange of individuals, for example individuals from differentpopulations. In addition, other bacterial homologues may be obtained andsuch homologues and fragments thereof in general will be capable ofselectively hybridising to the sequences shown in the sequence listingherein.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the present invention. Conservedsequences can be predicted, for example, by aligning the amino acidsequences from several variants/homologues. Sequence alignments can beperformed using computer software known in the art. For example the GCGWisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degeneratepositions and will be used at stringency conditions lower than thoseused for cloning sequences with single sequence primers against knownsequences.

Polynucleotides of the invention may be used to produce a primer, e.g. aPCR primer, a primer for an alternative amplification reaction, a probee.g. labelled with a revealing label by conventional means usingradioactive or non-radioactive labels, or the polynucleotides may becloned into vectors. Such primers, probes and other fragments will be atleast 15, preferably at least 20, for example at least 25, 30 or 40nucleotides in length, and are also encompassed by the termpolynucleotides of the invention as used herein. Preferred fragments areless than 5000, 2000, 1000, 500 or 200 nucleotides in length.

Polynucleotides such as a DNA polynucleotides and probes according tothe invention may be produced recombinantly, synthetically, or by anymeans available to those of skill in the art. They may also be cloned bystandard techniques.

In general, primers will be produced by synthetic means, involving astep wise manufacture of the desired nucleic acid sequence onenucleotide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about 15to 30 nucleotides) flanking a region of the sequence which it is desiredto clone, bringing the primers into contact with mRNA or cDNA obtainedfrom an animal or human cell, performing a polymerase chain reactionunder conditions which bring about amplification of the desired region,isolating the amplified fragment (e.g. by purifying the reaction mixtureon an agarose gel) and recovering the amplified DNA. The primers may bedesigned to contain suitable restriction enzyme recognition sites sothat the amplified DNA can be cloned into a suitable cloning vector.

Vectors, Host Cells, Expression Systems

The invention may employ vectors that comprise a polynucleotide whichcodes for at least a chimeric Imp or MsbA protein or may comprisepolynucleotides of the present invention which code for a mutant Imp orMsbA protein with reduced LPS transporter activity of the presentinvention. Host cells that are genetically engineered with vectors ofthe invention (which may alter the genome of the cell) and theproduction of mutant, preferably chimeric Imp proteins by recombinanttechniques are further aspects of the invention. Cell-free translationsystems can also be employed to produce such proteins using RNAs derivedfrom DNA constructs.

Recombinant proteins of the present invention may be prepared byprocesses well known to those skilled in the art from geneticallyengineered host cells comprising expression systems.

For recombinant production of the proteins of the invention, host cellscan be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis, et al., BASICMETHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as cells of streptococci, staphylococci, enterococci, E. coli,streptomyces, cyanobacteria, Bacillus subtilis, Moraxella catarrhalis,Haemophilus influenzae and Neisseria meningitidis; fungal cells, such ascells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candidaalbicans and Aspergillus; insect cells such as cells of Drosophila S2and Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK,293, CV-1 and Bowes melanoma cells; and plant cells, such as cells of agymnosperm or angiosperm.

A great variety of expression systems can be used to produce theproteins of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picornaviruses, retroviruses, and alphaviruses and vectorsderived from combinations thereof, such as those derived from plasmidand bacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may contain control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express aprotein in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed protein. Thesesignals may be endogenous to the protein or they may be heterologoussignals. Proteins of the present invention can be recovered and purifiedfrom recombinant cell cultures by the method of the present invention.

Antibodies

The proteins of the invention can be used as immunogens to produceantibodies immunospecific for such proteins.

In certain preferred embodiments of the invention there are providedantibodies against the Imp or MsbA protein of the invention.

Antibodies generated against the proteins of the invention can beobtained by administering the proteins of the invention, orepitope-bearing fragments of either or both, analogues of either orboth, to an animal, preferably a nonhuman, using routine protocols. Forpreparation of monoclonal antibodies, any technique known in the artthat provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256:495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies to proteinsof this invention. Also, transgenic mice, or other organisms or animals,such as other mammals, may be used to express humanized antibodiesimmunospecific to the proteins of the invention.

Alternatively, phage display technology may be utilized to selectantibody genes with binding activities towards a protein of theinvention either from repertoires of PCR amplified v-genes oflymphocytes from humans screened for possessing anti-FrpB or from naivelibraries (McCafferty, et al., (1990), Nature 348, 552-554; Marks, etal., (1992) Biotechnology 10, 779-783). The affinity of these antibodiescan also be improved by, for example, chain shuffling (Clackson et al.,(1991) Nature 352: 628).

The above-described antibodies may be employed to isolate or to identifyclones expressing chimeric or mutated Imp or MsbA proteins of theinvention to purify the proteins or polynucleotides by, for example,affinity chromatography.

Thus, among others, antibodies against the Imp protein of the inventionmay be employed to treat infections, particularly bacterial infections,preferably Neisserial infections.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized,” where thecomplimentarity determining region or regions of the hybridoma-derivedantibody has been transplanted into a human monoclonal antibody, forexample as described in Jones et al (1986), Nature 321, 522-525 orTempest et al, (1991) Biotechnology 9, 266-273.

A protein of the present invention can be administered to a recipientwho then acts as a source of immune globulin, produced in response tochallenge from the specific vaccine. A subject thus treated would donateplasma from which hyperimmune globulin would be obtained viaconventional plasma fractionation methodology. The hyperimmune globulinwould be administered to another subject in order to impart resistanceagainst or treat Neisserial infection. Hyperimmune globulins of theinvention are particularly useful for treatment or prevention ofNeisserial disease in infants, immune compromised individuals or wheretreatment is required and there is no time for the individual to produceantibodies in response to vaccination.

An additional aspect of the invention is a pharmaceutical compositioncomprising a monoclonal antibody (or fragments thereof; preferably humanor humanised) reactive against the pharmaceutical composition of theinvention, which could be used to treat or prevent infection by Gramnegative bacteria, preferably Neisseria, more preferably Neisseriameningitidis or Neisseria gonorrhoeae and most preferably Neisseriameningitidis serogroup B.

Such pharmaceutical compositions comprise monoclonal antibodies that canbe whole immunoglobulins of any class e.g. IgG1-4, IgM, IgA1 or 2, IgDor IgE, chimeric antibodies or hybrid antibodies with specificity to twoor more antigens of the invention. They may also be fragments e.g.F(ab′)2, Fab′, Fab, Fv, ScFv and the like including hybrid fragments.

Methods of making monoclonal antibodies are well known in the art andcan include the fusion of splenocytes with myeloma cells (Kohler andMilstein 1975 Nature 256; 495; Antibodies—a laboratory manual Harlow andLane 1988). Alternatively, monoclonal Fv fragments can be obtained byscreening a suitable phage display library (Vaughan T J et al 1998Nature Biotechnology 16; 535). Monoclonal antibodies may be humanised orpart humanised by known methods.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal,preferably humans, which comprises inoculating the individual with theGram negative bacterium of the invention or a fraction or membranethereof, or with the chimeric protein of the invention or with an outermembrane vesicle of the invention or a pharmaceutical composition orvaccine of the invention, adequate to produce antibody and/ or T cellimmune response to protect (or treat) said individual from infection,particularly bacterial infection and most particularly Neisseriameningitidis infection. Also provided are methods whereby suchimmunological response slows bacterial replication.

A further aspect of the invention relates to a pharmaceuticalcomposition or vaccine that when introduced into an individual,preferably a human, capable of having induced within it an immunologicalresponse, induces an immunological response in such individual to achimeric protein of the present invention. Preferably the immunologicalresponse is against an Imp epitope and at least one insert epitope froma separate protein. The immunological response may be usedtherapeutically or prophylactically and may take the form of antibodyimmunity and/or cellular immunity, such as cellular immunity arisingfrom CTL or CD4+ T cells.

Also provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the proteins of the invention andimmunostimulatory DNA sequences, such as those described in Sato, Y. etal. Science 273: 352 (1996).

The invention thus also includes a vaccine formulation which comprises aGram negative bacterium of the present invention or fraction thereof, ora chimeric protein of the present invention or an outer membrane vesiclepreparation of the invention, together with a suitable carrier, such asa pharmaceutically acceptable carrier. Since the proteins may be brokendown in the stomach, each is preferably administered parenterally,including, for example, administration that is subcutaneous,intramuscular, intravenous, or intradermal. Formulations suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions which may contain anti-oxidants, buffers,bacteristatic compounds and solutes which render the formulationisotonic with the bodily fluid, preferably the blood, of the individual;and aqueous and non-aqueous sterile suspensions which may includesuspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The formulation may also be administered mucosally, e.g.intranasally.

The vaccine formulation of the invention may also include adjuvantsystems for enhancing the immunogenicity of the formulation. Typicallyaluminium phosphate or aluminium hydroxide may be used. Preferably theadjuvant system raises preferentially a TH1 type of response.

An immune response may be broadly distinguished into two extremecategories, being a humoral or cell mediated immune responses(traditionally characterised by antibody and cellular effectormechanisms of protection respectively). These categories of responsehave been termed TH1-type responses (cell-mediated response), andTH2-type immune responses (humoral response).

Extreme TH1-type immune responses may be characterised by the generationof antigen specific, haplotype restricted cytotoxic T lymphocytes, andnatural killer cell responses. In mice TH1-type responses are oftencharacterised by the generation of antibodies of the IgG2a subtype,whilst in the human these correspond to IgG1 type antibodies. TH2-typeimmune responses are characterised by the generation of a broad range ofimmunoglobulin isotypes including in mice IgG1, IgA, and IgM.

It can be considered that the driving force behind the development ofthese two types of immune responses are cytokines. High levels ofTH1-type cytokines tend to favour the induction of cell mediated immuneresponses to the given antigen, whilst high levels of TH2-type cytokinestend to favour the induction of humoral immune responses to the antigen.

The distinction of TH1 and TH2-type immune responses is not absolute. Inreality an individual will support an immune response which is describedas being predominantly TH1 or predominantly TH2. However, it is oftenconvenient to consider the families of cytokines in terms of thatdescribed in murine CD4 +ve T cell clones by Mosmann and Coffman(Mosmann, T. R. and Coffman, R. L. (1989) TH1 and TH2 cells: differentpatterns of lymphokine secretion lead to different functionalproperties. Annual Review of Immunology, 7, p145-173). Traditionally,TH1-type responses are associated with the production of the INF-γ andIL-2 cytokines by T-lymphocytes. Other cytokines often directlyassociated with the induction of TH1-type immune responses are notproduced by T-cells, such as IL-12. In contrast, TH2-type responses areassociated with the secretion of IL-4, IL-5, IL-6 and IL-13.

It is known that certain vaccine adjuvants are particularly suited tothe stimulation of either TH1 or TH2 -type cytokine responses.Traditionally the best indicators of the TH1:TH2 balance of the immuneresponse after a vaccination or infection includes direct measurement ofthe production of TH1 or TH2 cytokines by T lymphocytes in vitro afterrestimulation with antigen, and/or the measurement of the IgG1:IgG2aratio of antigen specific antibody responses.

Thus, a TH1-type adjuvant is one which preferentially stimulatesisolated T-cell populations to produce high levels of TH1-type cytokineswhen re-stimulated with antigen in vitro, and promotes development ofboth CD8+cytotoxic T lymphocytes and antigen specific immunoglobulinresponses associated with TH1-type isotype.

Adjuvants which are capable of preferential stimulation of the TH1 cellresponse are described in International Patent Application No. WO94/00153 and WO 95/17209.

3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant,and is preferred. This is known from GB 2220211 (Ribi). Chemically it isa mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6acylated chains and is manufactured by Ribi Immunochem, Montana. Apreferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed inEuropean Patent 0 689 454 B1 (SmithKline Beecham Biologicals SA).Alternatively, other non-toxic derivatives of LPS may be used.

Preferably, the particles of 3D-MPL are small enough to be sterilefiltered through a 0.22 micron membrane (European Patent number 0 689454). 3D-MPL will be present in the range of 10 μg -100 μg preferably25-50 μg per dose wherein the antigen will typically be present in arange 2-50 μg per dose.

Another preferred adjuvant comprises QS21, an Hplc purified non-toxicfraction derived from the bark of Quillaja Saponaria Molina. Optionallythis may be admixed with 3 De-O-acylated monophosphoryl lipid A(3D-MPL), or other non-toxic LPS derivative, optionally together with acarrier.

The method of production of QS21 is disclosed in U.S. Pat. No.5,057,540.

Non-reactogenic adjuvant formulations containing QS21 have beendescribed previously (WO 96/33739). Such formulations comprising QS21and cholesterol have been shown to be successful TH1 stimulatingadjuvants when formulated together with an antigen.

Further adjuvants which are preferential stimulators of TH1 cellresponse include immunomodulatory oligonucleotides, for exampleunmethylated CpG sequences as disclosed in WO 96/02555.

Combinations of different TH1 stimulating adjuvants, such as thosementioned hereinabove, are also contemplated as providing an adjuvantwhich is a preferential stimulator of TH1 cell response. For example,QS21 can be formulated together with 3D-MPL. The ratio of QS21:3D-MPLwill typically be in the order of 1:10 to 10:1; preferably 1:5 to 5:1and often substantially 1:1. The preferred range for optimal synergy is2.5:1 to 1:1 3D-MPL:QS21.

Preferably a carrier is also present in the vaccine compositionaccording to the invention. The carrier may be an oil in water emulsion,or an aluminium salt, such as aluminium phosphate or aluminiumhydroxide.

A preferred oil-in-water emulsion comprises a metabolisible oil, such assqualene, alpha tocopherol and TWEEN® 80 (polysorbate 80). In aparticularly preferred aspect the antigens in the vaccine compositionaccording to the invention are combined with QS21 and 3D-MPL in such anemulsion. Additionally the oil in water emulsion may contain span 85and/or lecithin and/or tricaprylin.

Typically for human administration QS21 and 3D-MPL will be present in avaccine in the range of 1 μg -200 μg, such as 10-100 μg, preferably 10μg-50 μg per dose. Typically the oil in water will comprise from 2 to10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% TWEEN®80(polysorbate 80). Preferably the ratio of squalene: alpha tocopherolis equal to or less than 1 as this provides a more stable emulsion. Span85 may also be present at a level of 1%. In some cases it may beadvantageous that the vaccines of the present invention will furthercontain a stabiliser.

Non-toxic oil in water emulsions preferably contain a non-toxic oil,e.g. squalane or squalene, an emulsifier, e.g. TWEEN® 80 (polysorbate80), in an aqueous carrier. The aqueous carrier may be, for example,phosphate buffered saline.

A particularly potent adjuvant formulation involving QS21, 3D-MPL andtocopherol in oil in water emulsion is described in WO 95/17210.

The present invention also provides a polyvalent vaccine compositioncomprising a vaccine formulation of the invention in combination withother antigens, in particular antigens useful for treating cancers,autoimmune diseases and related conditions. Such a polyvalent vaccinecomposition may include a TH-1 inducing adjuvant as hereinbeforedescribed.

Outer Membrane Vesicle Preparations

A preferred embodiment of the invention is an outer membrane vesiclepreparation derived from the Gram negative bacterium of any one of theinvention or comprising the chimeric protein of the invention.

N. meningitidis serogroup B (menB) excretes outer membrane blebs insufficient quantities to allow their manufacture on an industrial scale.Outer membrane vesicles may also be prepared via the process ofdetergent extraction of the bacterial cells (see for example EP 11243).

The outer membrane vesicle preparation of the invention is therefore aconvenient way of presenting many antigens including Imp epitopes andepitopes from heterologous proteins within a context of other antigensfrom the Gram negative bacterium.

Preferably, the outer membrane vesicle preparation of the inventioncontains reduced levels of LPS due to the loss of LPS transportingactivity. Preferably, the presence of the chimeric protein of theinvention or an Imp and/or MsbA protein with reduced LPS transportingactivity results in a decrease of the amount of LPS on the outermembrane of at least 50%, 60%, 70%, preferably 80%, 90% or morepreferably 95% or 99% or 100% compared to an outer membrane vesiclepreparation derived from a similar strain of Gram negative bacteria inwhich Imp is not down regulated. This is preferably realised byisolating the outer membrane vesicles without a detergent extractionstep (or using less than or equal to 0.1, 0.05 or 0.01% DOC).

Most preferably, the outer membrane vesicle preparation of the inventioncontains a level of LPS sufficiently low so that the toxicity is reducedto a level at which the outer membrane vesicle preparation has anacceptable level of reactogenicity when inoculated into a patient.

Additional Features of the Outer Membrane Preparation

The outer membrane vesicle preparation has preferably been engineered tohave higher levels of expression of at additional antigens byrecombinantly upregulating their expression. Examples of antigens whichwould be upregulated in such a outer membrane vesicle preparation inaddition to the chimeric protein of the present invention include; NspA,Hsf, Hap, OMP85, TbpA (high), TbpA (low), LbpA, TbpB, LbpB, PilQ andPldA. Such preparations would optionally also comprise either or both ofLPS immunotype L2 and LPS immunotype L3.

The manufacture of bleb preparations from Neisserial strains may beachieved by any of the methods well known or apparent to a skilledperson. Preferably the methods disclosed in EP 301992, U.S. Pat. No.5,597,572, EP 11243 or U.S. Pat. No. 4,271,147, Frederikson et al. (NIPHAnnals [1991], 14:67-80), Zollinger et al. (J. Clin. Invest. [1979],63:836-848), Saunders et al. (Infect. Immun. [1999], 67:113-119),Drabick et al. (Vaccine [2000], 18:160-172) or WO 01/09350 (Example 8)are used. In general, OMVs are extracted with a detergent, preferablydeoxycholate, and nucleic acids are optionally removed enzymatically.Purification is achieved by ultracentrifugation optionally followed bysize exclusion chromatography. If 2 or more different blebs of theinvention are included, they may be combined in a single container toform a multivalent preparation of the invention (although a preparationis also considered multivalent if the different blebs of the inventionare separate compositions in separate containers which are administeredat the same time [the same visit to a practitioner] to a host). OMVpreparations are usually sterilised by filtration through a 0.2 μmfilter, and are preferably stored in a sucrose solution (e.g. 3%) whichis known to stabilise the bleb preparations.

Upregulation of proteins within outer membrane vesicle preparations maybe achieved by insertion of an extra copy of a gene into the Neisserialstrain from which the OMV preparation is derived. Alternatively, thepromoter of a gene can be exchanged for a stronger promoter in theNeisserial strain from which the OMV preparation is derived. Suchtechniques are described in WO01/09350. If an extra copy of the gene isintroduced, it too can have a non-native strong promoter operably linkedfor overexpression. Upregulation of a protein will lead to a higherlevel of protein being present in OMV compared to the level of proteinpresent in OMV derived from unmodified N. meningitidis (for instancestrain H44/76). Preferably the level will be 1.5, 2, 3, 4, 5, 7, 10 or20 times higher.

Where the presence of the chimeric protein of the invention does notlead to sufficiently low levels of LPS being present in the outermembrane vesicle preparation and LPS is intended to be an additionalantigen in the OMV, a protocol using a low concentration of extractingdetergent (for example deoxycholate or DOC) may preferably be used inthe OMV preparation method so as to preserve high levels of bound LPSwhilst removing particularly toxic, poorly bound LPS. The concentrationof DOC used is preferably 0-0.3% DOC, more preferably 0.05%-0.2% DOC,most preferably around 0.1% DOC.

“Stronger promoter sequence” refers to a regulatory control element thatincreases transcription for a gene encoding antigen of interest.

“Upregulating expression” refers to any means to enhance the expressionof an antigen of interest, relative to that of the non-modified (i.e.,naturally occurring) bleb. It is understood that the amount of‘upregulation’ will vary depending on the particular antigen of interestbut will not exceed an amount that will disrupt the membrane integrityof the bleb. Upregulation of an antigen refers to expression that is atleast 10% higher than that of the non-modified bleb. Preferably it is atleast 50% higher. More preferably it is at least 100% (2 fold) higher.Alternatively or additionally, upregulating expression may refer torendering expression non-conditional on metabolic or nutritionalchanges, particularly in the case of FrpB, TbpA, TbpB, LbpA and LbpB. Ingeneral where FrpB is overexpressed in a bleb this may be done byremoving regulatory sequences from the promoter, or by replacement ofthe promoter for a strong, non-regulated promoter such as PorA.

Again for the purpose of clarity, the terms ‘engineering a bacterialstrain to produce less of said antigen’ or down regulation refers to anymeans to reduce the expression of an antigen (or the expression of afunctional gene product) of interest, relative to that of thenon-modified (i.e., naturally occurring bleb), preferably by deletion,such that expression is at least 10% lower than that of the non-modifiedbleb. Preferably it is at least 50% lower and most preferably completelyabsent. If the down regulated protein is an enzyme or a functionalprotein, the downregulation may be achieved by introducing one or moremutations resulting in a 10%, 20%, 50%, 80% or preferably a 100%reduction in enzymatic or functional activity.

The engineering steps required to modulate the expression of Neisserialproteins can be carried out in a variety of ways known to the skilledperson. For instance, sequences (e.g. promoters or open reading frames)can be inserted, and promoters/genes can be disrupted by the techniqueof transposon insertion. For instance, for upregulating a gene'sexpression, a strong promoter could be inserted via a transposon up to 2kb upstream of the gene's initiation codon (more preferably 200-600 bpupstream, most preferably approximately 400 bp upstream). Point mutationor deletion may also be used (particularly for down-regulatingexpression of a gene).

Such methods, however, may be quite unstable or uncertain, and thereforeit is preferred that the engineering step is performed via a homologousrecombination event. Preferably, the event takes place between asequence (a recombinogenic region) of at least 30 nucleotides on thebacterial chromosome, and a sequence (a second recombinogenic region) ofat least 30 nucleotides on a vector transformed within the strain.Preferably the regions are 40-1000 nucleotides, more preferably 100-800nucleotides, most preferably 500 nucleotides). These recombinogenicregions should be sufficiently similar that they are capable ofhybridising to one another under highly stringent conditions.

Methods used to carry out the genetic modification events hereindescribed (such as the upregulation or downregulation of genes byrecombination events and the introduction of further gene sequences intoa Neisserial genome) are described in WO01/09350. Typical strongpromoters that may be integrated in Neisseria are porA, porB, IgtF, Opa,p110, Ist, and hpuAB. PorA and PorB are preferred as constitutive,strong promoters. It has been established that the PorB promoteractivity is contained in a fragment corresponding to nucleotides −1 to−250 upstream of the initiation codon of porB.

Down Regulation/Removal of Variable and Non-protective ImmunodominantAntigens

Many surface antigens are variable among bacterial strains and as aconsequence are protective only against a limited set of closely relatedstrains. An aspect of this invention covers outer membrane vesicles ofthe invention in which the expression of other proteins is reduced, or,preferably, gene(s) encoding variable surface protein(s) are deleted.Such deletion results in a bacterial strain producing blebs which, whenadministered in a vaccine, have a stronger potential forcross-reactivity against various strains due to a higher influenceexerted by conserved proteins (retained on the outer membranes) on thevaccinee's immune system. Examples of such variable antigens inNeisseria that may be downregulated in the bleb immunogenic compositionsof the invention include PorA, PorB, and Opa.

Other types of gene that could be down-regulated or switched off aregenes which, in vivo, can easily be switched on (expressed) or off bythe bacterium. As outer membrane proteins encoded by such genes are notalways present on the bacteria, the presence of such proteins in thebleb preparations can also be detrimental to the effectiveness of thevaccine for the reasons stated above. A preferred example todown-regulate or delete is Neisseria Opc protein. Anti-Opc immunityinduced by an Opc containing bleb vaccine would only have limitedprotective capacity as the infecting organism could easily become Opc⁻.

For example, these variable or non-protective genes may bedown-regulated in expression, or terminally switched off. This has theadvantage of concentrating the immune system on better antigens that arepresent in low amounts on the outer surface of blebs. By down-regulationit is also meant that surface exposed, variable immunodominant loops ofthe above outer membrane proteins may be altered or deleted in order tomake the resulting outer membrane protein less immunodominant.

Methods for downregulation of expression are disclosed in WO01/09350.Preferred combinations of proteins to be downregulated in the blebimmunogenic compositions of the invention include PorA and OpA; PorA andOpC; OpA and OpC; PorA and OpA and OpC.

Detoxification of LPS

In certain embodiments of the invention, where the outer membranevesicle preparation has too high a level of toxicity due to the presenceof LPS, the outer membrane vesicle preparation may be detoxified viamethods for detoxification of LPS which are disclosed in WO01/09350. Inparticular methods for detoxification of LPS of the invention involvethe downregulation of htrB and/or msbB enzymes are disclosed inWO01/09350. Such methods are preferably combined with methods of blebextraction involving low levels of DOC, preferably 0-0.3% DOC, morepreferably 0.05%-0.2% DOC, most preferably around 0.1% DOC.

Cross-reactive Polysaccharides

The isolation of bacterial outer-membrane blebs from encapsulatedGram-negative bacteria often results in the co-purification of capsularpolysaccharide. In some cases, this “contaminant” material may proveuseful since polysaccharide may enhance the immune response conferred byother bleb components. In other cases however, the presence ofcontaminating polysaccharide material in bacterial bleb preparations mayprove detrimental to the use of the blebs in a vaccine. For instance, ithas been shown at least in the case of N. meningitidis that theserogroup B capsular polysaccharide does not confer protective immunityand is susceptible to induce an adverse auto-immune response in humans.Consequently, outer membrane vesicles of the invention may be isolatedfrom a bacterial strain for bleb production, which has been engineeredsuch that it is free of capsular polysaccharide. The blebs will then besuitable for use in humans. A particularly preferred example of such ableb preparation is one from N. meningitidis serogroup B devoid ofcapsular polysaccharide.

This may be achieved by using modified bleb production strains in whichthe genes necessary for capsular biosynthesis and/or export have beenimpaired. Inactivation of the gene coding for capsular polysaccharidebiosynthesis or export can be achieved by mutating (point mutation,deletion or insertion) either the control region, the coding region orboth (preferably using the homologous recombination techniques describedabove), or by any other way of decreasing the enzymatic function of suchgenes. Moreover, inactivation of capsular biosynthesis genes may also beachieved by antisense over-expression or transposon mutagenesis. Apreferred method is the deletion of some or all of the Neisseriameningitidis cps genes required for polysaccharide biosynthesis andexport. For this purpose, the replacement plasmid pMF121 (described inFrosh et al. 1990, Mol. Microbiol. 4:1215-1218) can be used to deliver amutation deleting the cpsCAD (+galE) gene cluster.

Preferably the siaD gene is deleted, or down-regulated in expression orthe gene product enzymatically inactivated by any other way (themeningococcal siaD gene encodes alpha-2,3-sialyltransferase, an enzymerequired for capsular polysaccharide and LOS synthesis). This mutationis preferred in order to cause minimum disruption to LPS epitopes whichare preferably conserved in the preparations of the invention.

In bleb preparations, particularly in preparations extracted with lowDOC concentrations LPS may be used as an antigen in the immunogeniccomposition of the invention. It is however advantageous todownregulate/delete/inactivate enzymatic function of either the IgtE orpreferably IgtB genes/gene products in order to remove human likelacto-N-neotetraose structures. The Neisserial locus (and sequencethereof) comprising the Igt genes for the biosynthesis of LPSoligosaccharide structure is known in the art (Jennings et alMicrobiology 1999 145; 3013-3021). Downregulation/deletion of IgtB (orfunctional gene product) is preferred since it leaves the LPS protectiveepitope intact. In N. meningitidis serogroup B bleb preparations of theinvention, the downregulation/deletion of both siaD and IgtB ispreferred, leading to a bleb preparation with optimal safety and LPSprotective epitope retention.

Pharmaceutical compositions of the invention optionally comprise equalto or at least, one, two, three, four or five different outer membranevesicle preparations. Where two or more OMV preparations are included,at least one antigen is preferably upregulated in each OMV. Such OMVpreparations may be derived from Neisserial strains of the same speciesand serogroup or preferably from Neisserial strains of different class,serogroup, serotype, subserotype or immunotype. For example, animmunogenic composition may comprise one or more outer membrane vesiclepreparation(s) which contains LPS of immunotype L2 and one or more outermembrane vesicle preparation which contains LPS of immunotype L3. L2 orL3 OMV preparations are preferably derived from a stable strain whichhas minimal phase variability in the LPS oligosaccharide synthesis genelocus.

Combinations

The pharmaceutical compositions of the present invention, may alsocomprise at least one or more of the following:

-   a. one or more subunit vaccines;-   b. one or more outer membrane vesicles with one or more antigens    upregulated; and-   c. a mixture of a. and b.

The pharmaceutical compositions of the invention may thus also compriseboth a subunit composition and an outer membrane vesicle.

The outer membrane vesicle preparation may have at least one differentantigen selected from the following list which has been recombinantlyupregulated in an outer membrane vesicle: NspA, Hsf, Hap, OMP85, TbpA(high), TbpA (low), LbpA, TbpB, LbpB, NadA, TspA, TspB, PilQ and PldA;and optionally comprise either or both of LPS immunotype L2 and LPSimmunotype L3.

There are several antigens that are particularly suitable for inclusionin a subunit composition due to their solubility. Examples of suchproteins include; FhaB, NspA, passenger domain of Hsf, passenger domainof Hap, OMP85, FrpA, FrpC, TbpB, LbpB, PilQ.

Neisserial infections progress through several different stages. Forexample, the meningococcal life cycle involve nasopharyngealcolonisation, mucosal attachment, crossing into the bloodstream,multiplication in the blood, induction of toxic shock, crossing theblood/brain barrier and multiplication in the cerebrospinal fluid and/orthe meninges. Different molecules on the surface of the bacterium willbe involved in different steps of the infection cycle. By targeting theimmune response against an effective amount of a combination ofparticular antigens, involved in different processes of Neisserialinfection, a Neisserial vaccine with surprisingly high efficacy can beachieved.

In particular, combinations of certain Neisserial antigens fromdifferent classes with the chimeric protein of the invention can elicitan immune response which protects against multiple stages of infection.Such combinations of antigens can surprisingly lead to synergisticallyimproved vaccine efficacy against Neisserial infection where more thatone function of the bacterium is targeted by the immune response in anoptimal fashion. Some of the further antigens which can be included areinvolved in adhesion to host cells, some are involved in ironacquisition, some are autotransporters and some are toxins.

The efficacy of vaccines can be assessed through a variety of assays.Protection assays in animal models are well known in the art.Furthermore, serum bactericidal assay (SBA) is the most commonly agreedimmunological marker to estimate the efficacy of a meningococcal vaccine(Perkins et al. J Infect Dis. 1998, 177:683-691).

Some combinations of antigens can lead to improved protection in animalmodel assays and/or synergistically higher SBA titres. Without wishingto be bound by theory, such synergistic combinations of antigens areenabled by a number of characteristics of the immune response to theantigen combination. The antigens themselves are usually surface exposedon the Neisserial cells and tend to be conserved but also tend not to bepresent in sufficient quantity on the surface cell for an optimalbactericidal response to take place using antibodies elicited againstthe antigen alone. Combining the antigens of the invention can result ina formulation eliciting an advantageous combination of bactericidalantibodies which interact with the Neisserial cell beyond a criticalthreshold. At this critical level, sufficient antibodies of sufficientquality bind to the surface of the bacterium to allow efficient killingby complement and much higher bactericidal effects are seen as aconsequence. As serum bactericidal assays (SBA) closely reflect theefficacy of vaccine candidates, the attainment of good SBA titres by acombination of antigens is a good indication of the protective efficacyof a vaccine containing that combination of antigens.

An additional advantage of the invention is that the combination of theantigens of the invention from different families of proteins in animmunogenic composition will enable protection against a wider range ofstrains.

The invention thus also relates to immunogenic compositions comprising aplurality of proteins selected from at least two different categories ofprotein, having different functions within Neisseria. Examples of suchcategories of proteins are adhesins, autotransporter proteins, toxinsand Fe acquisition proteins. The vaccine combinations of the inventionshow surprising improvement in vaccine efficacy against homologousNeisserial strains (strains from which the antigens are derived) andpreferably also against heterologous Neisserial strains.

In particular, the invention provides immunogenic compositions thatcomprise at least one, two, three, four five, six, seven, eight, nine orten different additional Neisseria antigens (to FrpB) selected from atleast one, two, three, four or five groups of proteins selected from thefollowing:

-   at least one Neisserial adhesin selected from the group consisting    of FhaB, Hsf, NspA, NadA, PilC, Hap, MafA, MafB, Omp26, NMB0315,    NMB0995 and NMB1119;-   at least one Neisserial autotransporter selected from the group    consisting of Hsf, Hap, IgA protease, AspA and NadA;-   at least one Neisserial toxin selected from the group consisting of    FrpA, FrpC, FrpA/C, VapD, NM-ADPRT, and either or both of LPS    immunotype L2 and LPS immunotype L3;-   at least one Neisserial Fe acquisition protein selected from the    group consisting of TbpA high, TbpA low, TbpB high, TbpB low, LbpA,    LbpB, P2086, HpuA, HpuB, Lipo28, Sibp, FbpA, BfrA, BfrB, Bcp,    NMB0964 and NMB0293; and-   at least one Neisserial membrane associated protein, preferably    outer membrane protein, selected from the group consisting of PldA,    TspA, FhaC, NspA, TbpA (high), TbpA (low), LbpA, HpuB, TdfH, PorB,    HimD, HisD, GNA1870, OstA, HlpA, MltA, NMB 1124, NMB 1162, NMB 1220,    NMB 1313, NMB 1953, HtrA, TspB, PilQ and OMP85.    and preferably:-   a. at least one Neisserial adhesin selected from the group    consisting of FhaB, Hsf and NadA;-   b. at least one Neisserial autotransporter selected from the group    consisting of Hsf, Hap and NadA;-   c. at least one Neisserial toxin selected from the group consisting    of FrpA, FrpC, and either or both of LPS immunotype L2 and LPS    immunotype L3;-   d. at least one Neisserial Fe acquisition protein selected from the    group consisting of TbpA, TbpB, LbpA and LbpB; and-   e. at least one Neisserial outer membrane protein selected from the    group consisting of TspA, TspB, NspA, PilQ, OMP85, and PldA.

Preferably the first four (and most preferably all five) groups ofantigen are represented in the pharmaceutical composition of theinvention.

As previously mentioned where a protein is specifically mentionedherein, it is preferably a reference to a native, full-length proteinbut it may also encompass antigenic fragments thereof (particularly inthe context of subunit vaccines). These are fragments containing orcomprising at least 10 amino acids, preferably 20 amino acids, morepreferably 30 amino acids, more preferably 40 amino acids or mostpreferably 50 amino acids, taken contiguously from the amino acidsequence of the protein. In addition, antigenic fragments denotesfragments that are immunologically reactive with antibodies generatedagainst the Neisserial proteins or with antibodies generated byinfection of a mammalian host with Neisseria. Antigenic fragments alsoincludes fragments that when administered at an effective dose, elicit aprotective immune response against Neisserial infection, more preferablyit is protective against N. meningitidis and/or N. gonorrhoeaeinfection, most preferably it is protective against N. meningitidisserogroup B infection.

Also included in the invention are recombinant fusion proteins ofNeisserial proteins of the invention, or fragments thereof. These maycombine different Neisserial proteins or fragments thereof in the sameprotein. Alternatively, the invention also includes individual fusionproteins of Neisserial proteins or fragments thereof, as a fusionprotein with heterologous sequences such as a provider of T-cellepitopes, or viral surface proteins such as influenza virushaemagglutinin, tetanus toxoid, diphtheria toxoid, CRM197.

Addition Antigens of the Invention

NMB references refer to reference numbers to sequences which can beaccessed from www.neisseria.org.

1. Adhesins

Adhesins include FhaB (WO98/02547), NadA (J. Exp. Med (2002) 195:1445;NMB 1994), Hsf also known as NhhA (NMB 0992) (WO99/31132), Hap (NMB1985)(WO99/55873), NspA (WO96/29412), MafA (NMB 0652) and MafB (NMB0643) (Annu Rev Cell Dev Biol. 16; 423-457 (2000); Nature Biotech 20;914-921 (2002)), Omp26 (NMB 0181), NMB 0315, NMB 0995, NMB 1119 and PilC(Mol. Microbiol. 1997, 23; 879-892). These are proteins that areinvolved in the binding of Neisseria to the surface of host cells. Hsfis an example of an adhesin, as well as being an autotransporterprotein. Immunogenic compositions of the invention may therefore includecombinations of Hsf and other autotransporter proteins where Hsfcontributes in its capacity as an adhesin. These adhesins may be derivedfrom Neisseria meningitidis or Neisseria gonorrhoeae or other Neisserialstrains. The invention also includes other adhesins from Neisseria.

FhaB

This antigen has been described in WO98/02547 SEQ ID NO 38 (nucleotides3083-9025) —see also NMB0497. The present inventors have found FhaB tobe particularly effectively at inducing anti-adhesive antibodies aloneand in particular with other antigens of the invention. Although fulllength FhaB could be used, the inventors have found that particularC-terminal truncates are surprisingly at least as effective andpreferably even more effective in terms of cross-strain effect. Suchtruncates have also been advantageously shown to be far easier to clone.FhaB truncates of the invention typically correspond to the N-terminaltwo-thirds of the FhaB molecule, preferably the new C-terminus beingsituated at position 1200-1600, more preferably at position 1300-1500,and most preferably at position 1430-1440. Specific embodiments have theC-terminus at 1433 or 1436. Accordingly such FhaB truncates of theinvention and vaccines comprising such truncates are preferredcomponents of the combination immunogenic compositions of the invention.The N-terminus may also be truncated by up to 10, 20, 30, 40 or 50 aminoacids.

2. Autotransporter proteins

Autotransporter proteins typically are made up of a signal sequence, apassenger domain and an anchoring domain for attachment to the outermembrane. Examples of autotransporter proteins include Hsf (WO99/31132)(NMB 0992), HMW, Hia (van Ulsen et al Immunol. Med. Microbiol. 2001 32;53-64), Hap (NMB 1985) (WO99/55873; van Ulsen et al Immunol. Med.Microbiol. 2001 32; 53-64), UspA, UspA2, NadA (NMB 1994) (Comanducci etal J. Exp. Med. 2002 195; 1445-1454), AspA (Infection and Immunity 2002,70(8); 4447-4461; NMB 1029), Aida-1 like protein, SSh-2 and Tsh. NadA(J. Exp. Med (2002) 195:1445) is another example of an autotransporterproteins, as well as being an adhesin. Immunogenic compositions of theinvention may therefore include combinations of NadA and adhesins whereNadA contributes in its capacity as an autotransporter protein. Theseproteins may be derived from Neisseria meningitidis or Neisseriagonorrhoeae or other Neisserial strains. The invention also includesother autotransporter proteins from Neisseria.

Hsf

Hsf has a structure that is common to autotransporter proteins. Forexample, Hsf from N. meningitidis strain H44/76 consists of a signalsequence made up of amino acids 1-51, a head region at the aminoterminus of the mature protein (amino acids 52-479) that is surfaceexposed and contains variable regions (amino acids 52-106, 121-124,191-210 and 230-234), a neck region (amino acids 480-509), a hydrophobicalpha-helix region (amino acids 518-529) and an anchoring domain inwhich four transmembrane strands span the outer membrane (amino acids539-591).

Although full length Hsf may be used in immunogenic compositions of theinvention, various Hsf truncates and deletions may also beadvantageously used depending on the type of vaccine.

Where Hsf is used in a subunit vaccine, it is preferred that a portionof the soluble passenger domain is used; for instance the completedomain of amino acids 52 to 479, most preferably a conserved portionthereof, for instance the particularly advantageous sequence of aminoacids 134 to 479. Preferred forms of Hsf may be truncated so as todelete variable regions of the protein disclosed in WO01/55182.Preferred variants would include the deletion of one, two, three, four,or five variable regions as defined in WO01/55182. The above sequencesand those described below, can be extended or truncated by up to 1, 3,5, 7, 10 or 15 amino acids at either or both N or C termini.

Preferred fragments of Hsf therefore include the entire head region ofHsf, preferably containing amino acids 52-473. Additional preferredfragments of Hsf include surface exposed regions of the head includingone or more of the following amino acid sequences; 52-62, 76-93,116-134, 147-157, 157-175, 199-211, 230-252, 252-270, 284-306 328-338,362-391, 408-418, 430-440 and 469-479.

Where Hsf is present in an outer membrane vesicle preparation, it may beexpressed as the full-length protein or preferably as an advantageousvariant made up of a fusion of amino acids 1-51 and 134-591(yielding amature outer membrane protein of amino acid sequence 134 to theC-terminus). Preferred forms of Hsf may be truncated so as to deletevariable regions of the protein disclosed in W001155182. Preferredvariants would include the deletion of one, two, three, four, or fivevariable regions as defined in W001/55182. Preferably the first andsecond variable regions are deleted. Preferred variants would deleteresidues from between amino acid sequence 52 through to 237 or 54through to 237, more preferably deleting residues between amino acid 52through to 133 or 55 through to 133. The mature protein would lack thesignal peptide.

Hap

Computer analysis of the Hap-like protein from Neisseria meningitidisreveals at least three structural domains. Considering the Hap-likesequence from strain H44/76 as a reference, Domain 1, comprisingamino-acid 1 to 42, encodes a sec-dependant signal peptidecharacteristic of the auto-transporter family, Domain 2 comprisingamino-acids 43 to 950, encode the passenger domain likely to be surfaceexposed and accessible to the immune system, Domain 3, comprisingresidues 951 to the C-terminus (1457), is predicted to encode abeta-strands likely to assemble into a barrel-like structure and to beanchored into the outer-membrane. Since domains 2 is likely to besurface-exposed, well conserved (more than 80% in all strain tested) andcould be produced as subunit antigens in E. coli, it represents aninteresting vaccine candidates. Since domains 2 and 3 are likely to besurface-exposed, are well conserved (Pizza et al. (2000), Science 287:1816-1820), they represent interesting vaccine candidates. Domain 2 isknown as the passenger domain.

Immunogenic compositions of the invention may comprise the full-lengthHap protein, preferably incorporated into an OMV preparation.Immunogenic compositions of the invention may also comprise thepassenger domain of Hap which in strain H44/76 is composed of amino acidresidues 43-950. This fragment of Hap would be particularlyadvantageously used in a subunit composition of the invention. The abovesequence for the passenger domain of Hap can be extended or truncated byup to 1, 3, 5, 7, 10, 15, 20, 25, or 30 amino acids at either or both Nor C termini.

3. Iron Acquisition Proteins

Iron acquisition proteins include TbpA (NMB 0461) (WO92/03467, U.S. Pat.No. 5,912,336, WO93/06861 and EP586266), TbpB (NMB 0460) (WO93/06861 andEP586266), LbpA (NMB 1540) (Med Microbiol (1999) 32:1117), TbpB (NMB1541)(WO/99/09176), Hue (U73112.2) (Mol Microbiol. 1997, 23; 737-749),Hub (NC_(—)003116.1) (Mol Microbiol. 1997, 23; 737-749), P2086 alsoknown as XthA (NMB 0399) (13^(th) International Pathogenic NeisseriaConference 2002), FbpA (NMB 0634), FbpB, BfrA (NMB 1207), BfrB (NMB1206), Lipo28 also known as GNA2132 (NMB 2132), Sibp (NMB 1882), HmbR,HemH, Bcp (NMB 0750), Iron (III) ABC transporter-permease protein(Tettelin et al Science 287; 1809-1815 2000), Iron (III) ABCtransporter-periplasmic (Tettelin et al Science 287; 1809-1815 2000),TonB-dependent receptor (NMB 0964 and NMB 0293)(Tettelin et al Science287; 1809-1815 2000) and transferrin binding protein related protein(Tettelin et al Science 287; 1809-1815 2000). These proteins may bederived from Neisseria meningitidis, Neisseria gonorrhoeae or otherNeisserial strains. The invention also includes other iron acquisitionproteins from Neisseria.

TbpA

TbpA interacts with TbpB to form a protein complex on the outer membraneof Neisseria, which binds transferrin. Structurally, TbpA contains anintracellular N-terminal domain with a TonB box and plug domain,multiple transmembrane beta strands linked by short intracellular andlonger extracellular loops.

Two families of TbpB have been distinguished, having a high molecularweight and a low molecular weight respectively. High and low molecularweight forms of TbpB associate with different families of TbpA which aredistinguishable on the basis of homology. Despite being of similarmolecular weight, they are known as the high molecular weight and lowmolecular weight families because of their association with the high orlow molecular weight form of TbpB (Rokbi et al FEMS Microbiol. Lett.100; 51, 1993). The terms TbpA (high) and TbpA (low) are used to referto these two forms of TbpA, and similarly for TbpB. Immunogeniccompositions of the invention may comprise TbpA and

TbpB from serogroups A, B, C, Y and W-135 of N. meningitidis as well asiron acquisition proteins from other bacteria including N. gonorrhoeae.Transferrin binding proteins TbpA and TbpB have also been referred to asTbp1 and Tbp2 respectively (Cornelissen et al Infection and Immunity 65;822, 1997).

TbpA contains several distinct regions. For example, in the case of TbpAfrom N. meningitidis strain H44/76, the amino terminal 186 amino acidsform an internal globular domain, 22 beta strands span the membrane,forming a beta barrel structure. These are linked by short intracellularloops and larger extracellular loops. Extracellular loops 2, 3 and 5have the highest degree of sequence variability and loop 5 is surfaceexposed. Loops 5 and 4 are involved in ligand binding.

Preferred fragments of TbpA include the extracellular loops of TbpA.Using the sequence of TbpA from N. meningitidis strain H44/76, theseloops correspond to amino acids 200-202 for loop1, amino acids 226-303for loop 2, amino acids 348-395 for loop 3, amino acids 438-471 for loop4, amino acids 512-576 for loop 5, amino acids 609-625 for loop 6, aminoacids 661-671 for loop 7, amino acids 707-723 for loop 8, amino acids769-790 for loop 9, amino acids 814-844 for loop 10 and amino acids872-903 for loop 11. The corresponding sequences, after sequencealignment, in other Tbp proteins would also constitute preferredfragments. Most preferred fragments would include amino acid sequencesconstituting loop 2, loop 3, loop 4 or loop 5 of Tbp.

Where the immunogenic compositions of the invention comprise TbpA, it ispreferable to include both TbpA (high) and TbpA (low).

Although TbpA is preferably presented in an OMV vaccine, it may also bepart of a subunit vaccine. For instance, isolated iron acquisitionproteins which could be introduced into an immunogenic composition ofthe invention are well known in the art (WO00/25811). They may beexpressed in a bacterial host, extracted using detergent (for instance2% Elugent) and purified by affinity chromatography or using standardcolumn chromatography techniques well known to the art (Oakhill et alBiochem J. 2002 364; 613-6).

Where TbpA is presented in an OMV vaccine, its expression can beupregulated by genetic techniques discussed herein or in WO 01/09350, ormay preferably be upregulated by growth of the parent strain under ironlimitation conditions. This process will also result in the upregulationof variable iron-regulated proteins, particularly wild-type FrpB whichmay become immunodominant and it is therefore advantageous todownregulate the expression of (and preferably delete the genesencoding) such proteins (particularly wild-type FrpB) as described in WO01/09350, or remove its immunodominant loops as described above, toensure that the immunogenic composition of the invention elicits animmune response against antigens present in a wide range of Neisserialstrains. If wild-type FrpB is deleted, an additional copy of a nonimmunodominant mutant FrpB gene may be introduced into the cell. It ispreferred to have both TbpA(high) and TbpA(low) present in theimmunogenic composition and this is preferably achieved by combiningOMVs derived from two strains, expressing the alternative forms of TbpA.

4. Toxins

Toxins include FrpA (NMB 0585; NMB 1405), FrpA/C (see below fordefinition), FrpC (NMB 1415; NMB 1405) (WO92/01460), NM-ADPRT (NMB 1343)(13^(th) International Pathogenic Neisseria Conference 2002 Masignani etal p 135), VapD (NMB 1753), lipopolysaccharide (LPS; also calledlipooligosaccharide or LOS) immunotype L2 and LPS immunotype L3. FrpAand FrpC contain a region which is conserved between these two proteinsand a preferred fragment of the proteins would be a polypeptidecontaining this conserved fragment, preferably comprising amino acids227-1004 of the sequence of FrpA/C. These antigens may be derived fromNeisseria meningitidis or Neisseria gonorrhoeae or other Neisserialstrains. The invention also includes other toxins from Neisseria.

In an alternative embodiment, toxins may include antigens involved inthe regulation of toxicity, for example OstA which functions in thesynthesis of lipopolysaccharides.

FrpA and FrpC

Neisseria meningitidis encodes two RTX proteins, referred to as FrpA &FrpC secreted upon iron limitation (Thompson et al., (1993) J.Bacteriol. 175:811-818; Thompson et al., (1993) Infect. Immun.61:2906-2911). The RTX (Repeat ToXin) protein family have in common aseries of 9 amino acid repeat near their C-termini with the consensus:Leu Xaa Gly Gly Xaa Gly (Asn/Asp) Asp Xaa, SEQ ID NO: 16/SEQ ID NO:30.(LXGGXGN_(/D)DX). The repeats in E. coli HlyA are thought to be thesite of Ca2+ binding. As represented in FIG. 4, meningococcal FrpA andFrpC proteins, as characterized in strain FAM20, share extensiveamino-acid similarity in their central and C-terminal regions but verylimited similarity (if any) at the N-terminus. Moreover, the regionconserved between FrpA and FrpC exhibit some polymorphism due torepetition (13 times in FrpA and 43 times in FrpC) of a 9 amino acidmotif.

Immunogenic compositions of the invention may comprise the full lengthFrpA and/or FrpC or preferably, a fragment comprising the sequenceconserved between FrpA and FrpC. The conserved sequence is made up ofrepeat units of 9 amino acids. Immunogenic compositions of the inventionwould preferably comprise more that three repeats, more than 10 repeats,more than 13 repeats, more than 20 repeats or more than 23 repeats.

Such truncates have advantageous properties over the full lengthmolecules, and vaccines comprising such antigens are preferred for beingincorporated in the immunogenic compositions of the invention.

Sequences conserved between FrpA and FrpC are designated FrpA/C andwherever FrpA or FrpC forms a constituent of immunogenic compositions ofthe invention, FrpA/C could be advantageously used. Amino acids 277-1004of the FrpA sequence is the preferred conserved region. The abovesequence can be extended or truncated by up to 1, 3, 5, 7, 10, 15, 20,25, or 30 amino acids at either or both N or C termini.

LPS

LPS (lipopolysaccharide, also known as LOS—lipooligosaccharide) is theendotoxin on the outer membrane of Neisseria. The polysaccharide moietyof the LPS is known to induce bactericidal antibodies.

Heterogeneity within the oligosaccharide moiety of the LPS generatesstructural and antigenic diversity among different neisserial strains(Griffiss et al. Inf. Immun. 1987; 55: 1792-1800). This has been used tosubdivide meningococcal strains into 12 immunotypes (Scholtan et al. JMed Microbiol 1994, 41:236-243). Immunotypes L3, L7, & L9 areimmunologically identical and are structurally similar (or even thesame) and have therefore been designated L3,7,9 (or, for the purposes ofthis specification, generically as “L3”). Meningococcal LPS L3,7,9 (L3),L2 and L5 can be modified by sialylation, or by the addition of cytidine5′-monophosphate-N-acetyineuraminic acid. Although L2, L4 and L6 LPS aredistinguishable immunologically, they are structurally similar and whereL2 is mentioned herein, either L4 or L6 may be optionally substitutedwithin the scope of the invention. See M. P. Jennings et al,Microbiology 1999, 145, 3013-3021 and Mol Microbiol 2002, 43:931-43 forfurther illustration of LPS structure and heterogeneity.

Where LPS, preferably meningococcal LPS, is included in a vaccine of theinvention, preferably and advantageously either or both of immunotypesL2 and L3 are present. LPS is preferably presented in an outer membranevesicle (preferably where the vesicle is extracted with a low percentagedetergent, more preferably 0-0.5%, 0.02-0.4%, 0.04-0.3%, 0.06-0.2%,0.08-0.15% or 0.1%, most preferably deoxycholate [DOC]) but may also bepart of a subunit vaccine. LPS may be isolated using well knownprocedure including the hot water-phenol procedure (Wesphal and JannMeth. Carbo. Chem. 5; 83-91 1965). See also Galanos et al. 1969, Eur JBiochem 9:245-249, and Wu et al. 1987, Anal Bio Chem 160:281-289. LPSmay be used plain or conjugated to a source of T-cell epitopes such astetanus toxoid, Diphtheria toxoid, CRM-197 or OMV outer membraneproteins. Techniques for conjugating isolated LOS are also known (seefor instance EP 941738 incorporated by reference herein).

Where LOS (in particular the LOS of the invention) is present in a blebformulation the LOS is preferably conjugated in situ by methods allowingthe conjugation of LOS to one or more outer membrane proteins alsopresent on the bleb preparation (e.g. PorA or PorB in meningococcus).

This process can advantageously enhance the stability and/orimmunogenicity (providing T-cell help) and/or antigenicity of the LOSantigen within the bleb formulation—thus giving T-cell help for theT-independent oligosaccharide immunogen in its most protectiveconformation—as LOS in its natural environment on the surface ofmeningococcal outer membrane. In addition, conjugation of the LOS withinthe bleb can result in a detoxification of the LOS (the Lipid A portionbeing stably buried in the outer membrane thus being less available tocause toxicity). Thus the detoxification methods mentioned herein ofisolating blebs from htrB⁻ or msbB⁻ mutants, or by adding non toxicpeptide functional equivalent of polymyxin B [a molecule with highaffinity to Lipid A] to the composition (see WO 93/14115, WO 95/03327,Velucchi et al (1997) J Endotoxin Res 4: 1-12, and EP 976402 for furtherdetails of non-toxic peptide functional equivalents of polymyxinB—particularly the use of the peptide SAEP 2 (of sequence KTKCKFLKKC,SEQ ID NO: 17, where the 2 cysteines form a disulphide bridge)) may notbe required (but which may be added in combination for additionalsecurity). Thus the inventors have found that a composition comprisingblebs wherein LOS present in the blebs has been conjugated in anintra-bleb fashion to outer membrane proteins also present in the blebcan form the basis of a vaccine for the treatment or prevention ofdiseases caused by the organism from which the blebs have been derived,wherein such vaccine is substantially non-toxic and is capable ofinducing a T-dependent bactericidal response against LOS in its nativeenvironment.

Such bleb preparations may be isolated from the bacterial in question(see WO 01/09350), and then subjected to known conjugation chemistriesto link groups (e.g. NH₂ or COOH) on the oligosaccharide portion of LOSto groups (e.g. NH₂ or COOH) on bleb outer membrane proteins.Cross-linking techniques using glutaraldehyde, formaldehyde, orglutaraldehyde/formaldehyde mixes may be used, but it is preferred thatmore selective chemistries are used such as EDAC or EDAC/NHS (J. V.Staros, R. W. Wright and D. M. Swingle. Enhancement byN-hydroxysuccinimide of water-soluble carbodiimide-mediated couplingreactions. Analytical chemistry 156: 220-222 (1986); and BioconjugatesTechniques. Greg T. Hermanson (1996) pp 173-176). Other conjugationchemistries or treatments capable of creating covalent links between LOSand protein molecules that could be used are described in EP 941738.

Preferably the bleb preparations are conjugated in the absence ofcapsular polysaccharide. The blebs may be isolated from a strain whichdoes not produce capsular polysaccharide (naturally or via mutation asdescribed below), or may be purified from most and preferably allcontaminating capsular polysaccharide. In this way, the intra-bleb LOSconjugation reaction is much more efficient.

Preferably more than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of theLOS present in the blebs is cross-linked/conjugated.

Intrableb conjugation should preferably incorporate 1, 2 or all 3 of thefollowing process steps: conjugation pH should be greater than pH 7.0,preferably greater than or equal to pH 7.5 (most preferably under pH 9);conditions of 1-5% preferably 2-4% most preferably around 3% sucroseshould be maintained during the reaction; NaCl should be minimised inthe conjugation reaction, preferably under 0.1M, 0.05M, 0.01M, 0.005M,0.001M, and most preferably not present at all. All these processfeatures make sure that the blebs remain stable and in solutionthroughout the conjugation process.

The EDAC/NHS conjugation process is a preferred process for intra-blebconjugation. EDACINHS is preferred to formaldehyde which can cross-linkto too high an extent thus adversely affecting filterability. EDACreacts with carboxylic acids (such as KDO in LOS) to create anactive-ester intermediate. In the presence of an amine nucleophile (suchas lysines in outer membrane proteins such as PorB), an amide bond isformed with release of an isourea by-product. However, the efficiency ofan EDAC-mediated reaction may be increased through the formation of aSulfo-NHS ester intermediate. The Sulfo-NHS ester survives in aqueoussolution longer than the active ester formed from the reaction of EDACalone with a carboxylate. Thus, higher yields of amide bond formationmay be realized using this two-stage process. EDAC/NHS conjugation isdiscussed in J. V. Staros, R. W. Wright and D. M. Swingle. Enhancementby N-hydroxysuccinimide of water-soluble carbodiimide-mediated couplingreactions. Analytical chemistry 156: 220-222 (1986); and BioconjugatesTechniques. Greg T. Hermanson (1996) pp 173-176. Preferably 0.01-5 mgEDAC / mg bleb is used in the reaction, more preferably 0.05-1 mgEDAC/mg bleb. The amount of EDAC used depends on the amount of LOSpresent in the sample which in turn depends on the deoxycholate (DOC) %used to extract the blebs. At low % DOC (e.g. 0.1%), high amounts ofEDAC are used (1 mg/mg and beyond), however at higher % DOC (e.g. 0.5%),lower amounts of EDAC are used (0.025-0.1 mg/mg) to avoid too muchinter-bleb crosslinking.

A preferred process of the invention is therefore a process forproducing intra-bleb conjugated LOS (preferably meningococcal)comprising the steps of conjugating blebs in the presence of EDAC/NHS ata pH between pH 7.0 and pH 9.0 (preferably around pH 7.5), in 1-5%(preferably around 3%) sucrose, and optionally in conditionssubstantially devoid of NaCl (as described above), and isolating theconjugated blebs from the reaction mix.

The reaction may be followed on Western separation gels of the reactionmixture using anti-LOS (e.g. anti-L2 or anti-L3) mAbs to show theincrease of LOS molecular weight for a greater proportion of the LOS inthe blebs as reaction time goes on.

Yields of 99% blebs can be recovered using such techniques.

EDAC was found to be an excellent intra-bleb cross-linking agent in thatit cross-linked LOS to OMP sufficiently for improved LOS T-dependentimmunogenicity, but did not cross link it to such a high degree thatproblems such as poor filterability, aggregation and inter-blebcross-linking occurred. The morphology of the blebs generated is similarto that of unconjugated blebs (by electron microscope). In addition, theabove protocol avoided an overly high cross-linking to take place (whichcan decrease the immunogenicity of protective OMPs naturally present onthe surface of the bleb e.g. TbpA or Hsf).

It is preferred that the meningococcal strain from which the blebs arederived is a mutant strain that cannot produce capsular polysaccharide(in particular saiD⁻). It is also preferred that immunogeniccompositions effective against meningococcal disease comprise both an L2and L3 bleb, wherein the L2 and L3 LOS are both conjugated to bleb outermembrane proteins. Furthermore, it is preferred that the LOS structurewithin the intra-bleb conjugated bleb is consistent with it having beenderived from an IgtE⁻ or, preferably, IgtB⁻ meningococcal strain. Mostpreferably immunogenic compositions comprise intrableb-conjugated blebs:derived from a mutant meningococcal strain that cannot produce capsularpolysaccharide and is IgtB⁻; comprising L2 and L3 blebs derived frommutant meningococcal strains that cannot produce capsularpolysaccharide; comprising L2 and L3 blebs derived from mutantmeningococcal strains that are IgtB⁻; or most preferably comprising L2and L3 blebs derived from mutant meningococcal strains that cannotproduce capsular polysaccharide and are IgtB⁻.

Typical L3 meningococcal strain that can be used for the presentinvention is H44/76 menB strain. A typical L2 strain is the B16B6 menBstrain or the 39E meningococcus type C strain.

As stated above, the blebs of the invention have been detoxified to adegree by the act of conjugation, and need not be detoxified anyfurther, however further detoxification methods may be used foradditional security, for instance using blebs derived from ameningococcal strain that is htrB⁻ or msbB⁻ or adding a non-toxicpeptide functional equivalent of polymyxin B [a molecule with highaffinity to Lipid A] (preferably SAEP 2) to the bleb composition (asdescribed above).

In the above way meningococcal blebs and immunogenic compositionscomprising blebs are provided which have as an important antigen LOSwhich is substantially non-toxic, devoid of autoimmunity problems, has aT-dependent character, is present in its natural environment, and iscapable of inducing a bactericidal antibody response against more than90% of meningococcal strains (in the case of L2+L3 compositions).

5. Integral Outer Membrane Proteins

Other categories of Neisserial proteins may also be candidates forinclusion in the Neisserial vaccines of the invention and may be able tocombine with other antigens in a surprisingly effective manner. Membraneassociated proteins, particularly integral membrane proteins and mostadvantageously outer membrane proteins, especially integral outermembrane proteins may be used in the compositions of the presentinvention. An example of such a protein is PldA also known as Omp1A (NMB0464) (WO00/15801) which is a Neisserial phospholipase outer membraneprotein. Further examples are TspA (NMB 0341) (Infect. Immun. 1999, 67;3533-3541) and TspB (T-cell stimulating protein) (WO 00/03003; NMB 1548,NMB 1628 or NMB 1747). Further examples include PilQ (NMB 1812)(WO99/61620), OMP85 —also known as D15- (NMB 0182) (WO00/23593), NspA(U52066) (WO96/29412), FhaC (NMB 0496 or NMB 1780), PorB (NMB 2039)(Mol. Biol. Evol. 12; 363-370, 1995), HpuB (NC_(—)003116.1), TdfH (NMB1497) (Microbiology 2001, 147; 1277-1290), OstA (NMB 0280), MltA alsoknown as GNA33 and Lipo3O (NMB0033), HtrA (NMB 0532; WO 99/55872), HimD(NMB 1302), HisD (NMB 1581), GNA 1870 (NMB 1870), HIpA (NMB 1946), NMB1124, NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, TbpA (NMB 0461)(W092/03467) (see also above under iron acquisition proteins) and LbpA(NMB 1541).

OMP85

OMP85/D15 is an outer membrane protein having a signal sequence, aN-terminal surface-exposed domain and an integral membrane domain forattachment to the outer membrane. Immunogenic compositions of theinvention may also comprise the full length OMP85, preferably as part ofan OMV preparation. Fragments of OMP85 may also be used in immunogeniccompositions of the invention, in particularly, the N terminalsurface-exposed domain of OMP85 made up of amino acid residues 1-475 or50-475 is preferably incorporated into a subunit component of theimmunogenic compositions of the invention. The above sequence for the Nterminal surface-exposed domain of OMP85 can be extended or truncated byup to 1, 3, 5, 7, 10, 15, 20, 25, or 30 amino acids at either or both Nor C termini. It is preferred that the signal sequence is omitted fromthe OMP85 fragment.

OstA

OstA functions in the transport of lipopolysaccharides and may beconsidered to be a regulator of toxicity. OstA is optionally included inthe toxin category where the toxin category is broadened to containregulators of toxicity as well as toxins.

Preferably the subunit composition comprises a chimeric Imp/OstA proteinof the present invention together with:

-   i) at least one further antigen selected from the following list:    FhaB, passenger domain of Hsf, passenger domain of Hap, NadA,    N-terminal surface exposed domain of OMP85, FrpA, FrpC, FrpA/C,    TpbA, TbpB, LpbA, LbpB, PldA, PIIQ, NspA and either or both of LPS    immunotype L2 and LPS immunotype L3; and/or-   ii) at least a Neisserial (preferably meningococcal) outer membrane    vesicle (OMV) preparation. Preferably the OMV preparation has at    least one antigen (more preferably 2, 3, 4 or 5) selected from the    following list which has been recombinantly upregulated in the outer    membrane vesicle: FhaB, Hsf, NspA, NadA, PilC, Hap, MafA, MafB,    Omp26, NMB0315, NMB0995, NMB1119, IgA protease, AspA, TbpA high,    TbpA low, TbpB high, TbpB low, LbpA, LbpB, P2086, HpuA, HpuB,    Lipo28, Sibp, FbpA, BfrA, BfrB, Bcp, NMB0964 and NMB0293

When i) is present the additional antigen is preferably selected fromone or more of the groups of proteins given above.

In another embodiment the outer membrane vesicle of the presentinvention has at least one further antigen (more preferably 2, 3, 4 or5) is recombinantly upregulated in the outer membrane vesicle andselected from the following list: NspA, Hsf, Hap, OMP85, TbpA (high),TbpA (low), LbpA, TbpB, LbpB, PilQ and PldA; and optionally comprisingeither or both of LPS immunotype L2 and LPS immunotype L3. This outermembrane vesicle may be used with one or more further outer membranevesicles in which has at least one further antigen (more preferably 2,3, 4 or 5) is recombinantly upregulated in the outer membrane vesicleand selected from the following list: FrpB, NspA, Hsf, Hap, OMP85, TbpA(high), TbpA (low), LbpA, TbpB, LbpB, PilQ and PldA; and optionallycomprising either or both of LPS immunotype L2 and LPS immunotype L3.

The immunogenic compositions of the invention may comprise antigens(proteins, LPS and polysaccharides) derived from Neisseria meningitidisserogroups A, B, C, Y, W-135 or Neisseria gonorrhoeae.

Further Combinations

The pharmaceutical composition of the invention may further comprisebacterial capsular polysaccharides or oligosaccharides. The capsularpolysaccharides or oligosaccharides may be derived from one or more of:Neisseria meningitidis serogroup A, C, Y, and/or W-135, Haemophilusinfluenzae b, Streptococcus pneumoniae, Group A Streptococci, Group BStreptococci, Staphylococcus aureus and Staphylococcus epidermidis.

A further aspect of the invention are vaccine combinations comprisingthe antigenic composition of the invention with other antigens which areadvantageously used against certain disease states including thoseassociated with viral or Gram positive bacteria.

In one preferred combination, the pharmaceutical compositions of theinvention are formulated with 1, 2, 3 or preferably all 4 of thefollowing meningococcal capsular polysaccharides or oligosaccharideswhich may be plain or conjugated to a protein carrier: A, C, Y or W-135.Preferably the immunogenic compositions of the invention are formulatedwith A and C; or C; or C and Y. Such a vaccine containing proteins fromN. meningitidis serogroup B may be advantageously used as a globalmeningococcus vaccine.

In a further preferred embodiment, the pharmaceutical compositions ofthe invention, preferably formulated with 1, 2, 3 or all 4 of the plainor conjugated meningococcal capsular polysaccharides or oligosaccharidesA, C, Y or W-135 (as described above), are formulated with a conjugatedH. influenzae b capsular polysaccharide (or oligosaccharides), and/orone or more plain or conjugated pneumococcal capsular polysaccharides(or oligosaccharides) (for instance those described below). Optionally,the vaccine may also comprise one or more protein antigens that canprotect a host against Streptococcus pneumoniae infection. Such avaccine may be advantageously used as a global meningitis vaccine.

In a still further preferred embodiment, the pharmaceutical compositionof the invention is formulated with capsular polysaccharides oroligosaccharides derived from one or more of Neisseria meningitidis,Haemophilus influenzae b, Streptococcus pneumoniae, Group AStreptococci, Group B Streptococci, Staphylococcus aureus orStaphylococcus epidermidis. The pneumococcal capsular polysaccharide oroligosaccharide antigens are preferably selected from serotypes 1, 2, 3,4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F (most preferably from serotypes 1, 3, 4, 5, 6B, 7F, 9V,14, 18C, 19F and 23F). A further preferred embodiment would contain thePRP capsular polysaccharides or oligosaccharides of Haemophilusinfluenzae. A further preferred embodiment would contain the Type 5,Type 8 or 336 capsular polysaccharides of Staphylococcus aureus. Afurther preferred embodiment would contain the Type I, Type II or TypeIII capsular polysaccharides of Staphylococcus epidermidis. A furtherpreferred embodiment would contain the Type Ia, Type Ic, Type II or TypeIII capsular polysaccharides of Group B streptococcus. A furtherpreferred embodiment would contain the capsular polysaccharides of GroupA streptococcus, preferably further comprising at least one M proteinand more preferably multiple types of M protein.

Such capsular polysaccharides or oligosaccharides of the invention maybe unconjugated or conjugated to a carrier protein such as tetanustoxoid, tetanus toxoid fragment C, diphtheria toxoid, CRM197,pneumolysin, Protein D (U.S. Pat. No. 6,342,224). The polysaccharide oroligosaccharide conjugate may be prepared by any known couplingtechnique. For example the polysaccharide can be coupled via a thioetherlinkage. This conjugation method relies on activation of thepolysaccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP) to form a cyanate ester. The activated polysaccharide may thus becoupled directly or via a spacer group to an amino group on the carrierprotein. Preferably, the cyanate ester is coupled with hexane diamineand the amino-derivatised polysaccharide is conjugated to the carrierprotein using heteroligation chemistry involving the formation of thethioether linkage. Such conjugates are described in PCT publishedapplication WO93/15760 Uniformed Services University.

The conjugates can also be prepared by direct reductive aminationmethods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat.No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188,EP-208375 and EP-0-477508. A further method involves the coupling of acyanogen bromide activated polysaccharide derivatised with adipic acidhydrazide (ADH) to the protein carrier by Carbodiimide condensation (ChuC. et al Infect. Immunity, 1983 245 256).

Preferred pneumococcal proteins antigens are those pneumococcal proteinswhich are exposed on the outer surface of the pneumococcus (capable ofbeing recognised by a host's immune system during at least part of thelife cycle of the pneumococcus), or are proteins which are secreted orreleased by the pneumococcus. Most preferably, the protein is a toxin,adhesin, 2-component signal tranducer, or lipoprotein of Streptococcuspneumoniae, or fragments thereof. Particularly preferred proteinsinclude, but are not limited to: pneumolysin (preferably detoxified bychemical treatment or mutation) [Mitchell et al. Nucleic Acids Res. 1990Jul. 11; 18(13): 4010 “Comparison of pneumolysin genes and proteins fromStreptococcus pneumoniae types 1 and 2.”, Mitchell et al. BiochimBiophys Acta 1989 Jan 23; 1007(1): 67-72 “Expression of the pneumolysingene in Escherichia coli: rapid purification and biologicalproperties.”, WO 96/05859 (A. Cyanamid), WO 90/06951 (Paton et al), WO99/03884 (NAVA)]; PspA and transmembrane deletion variants thereof (U.S.Pat. No. 5,804,193 —Briles et al.); PspC and transmembrane deletionvariants thereof (WO 97/09994 —Briles et al); PsaA and transmembranedeletion variants thereof (Berry & Paton, Infect Immun 1996 December;64(12):5255-62 “Sequence heterogeneity of PsaA, a 37-kilodalton putativeadhesin essential for virulence of Streptococcus pneumoniae”);pneumococcal choline binding proteins and transmembrane deletionvariants thereof; CbpA and transmembrane deletion variants thereof (WO97/41151; WO 99/51266); Glyceraldehyde-3-phosphate -dehydrogenase(Infect. Immun. 1996 64:3544); HSP70 (WO 96140928); PcpA (Sanchez-Beatoet al. FEMS Microbiol Lett 1998, 164:207-14); M like protein, (EP0837130) and adhesin 18627, (EP 0834568). Further preferred pneumococcalprotein antigens are those disclosed in WO 98/18931, particularly thoseselected in WO 98/18930 and PCT/US99/30390.

The pharmaceutical composition/vaccine of the invention may alsooptionally comprise outer membrane vesicle preparations made from otherGram negative bacteria, for example Moraxella catarrhalis or Haemophilusinfluenzae.

Compositions, Kits and Administration

A vaccine is a composition comprising at least one antigen which iscapable of generating an immune response when administered to a host.Preferably, such vaccines are capable of generating a protective immuneresponse against Neisserial, preferably Neisseria meningitidis and/orNeisseria gonorrhoeae infection.

The invention also relates to compositions comprising a Gram negativebacterium, a chimeric protein or an outer membrane vesicle preparationdiscussed herein. Such compositions of the invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to an individual. Such compositionscomprise, for instance, a media additive or a therapeutically effectiveamount of a protein of the invention and a pharmaceutically acceptablecarrier or excipient. Such carriers may include, but are not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol andcombinations thereof. The formulation should suit the mode ofadministration. The invention further relates to diagnostic andpharmaceutical packs and kits comprising one or more containers filledwith one or more of the ingredients of the aforementioned compositionsof the invention.

The pharmaceutical compositions of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

The composition will be adapted to the route of administration, forinstance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a protein or other compounds of the present invention canbe formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, solutions, powders and the like.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

The dosage range required depends on the choice of peptide, the route ofadministration, the nature of the formulation, the nature of thesubject's condition, and the judgement of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

Preferred features and embodiment of the present invention will now bedescribed further with reference to the following non-limiting Examples:

EXAMPLE 1 General Methods

Bacterial Strains and Growth Conditions. Neisseria meningitidis (Nme)H44/76, a serotype B strain, came from our laboratory collection. TheH44/76 IpxA mutant (Steeghs et al 1998; Nature 392; 449-450) and theH44/76 derived strain HA3003, where IpxA expression is controlled by thetac promoter (Steeghs et al 2001; EMBO J. 24; 6937-6945), weregenerously provided by L. Steeghs and P. van der Ley (NetherlandsVaccine Institute (NVI), Bilthoven, The Netherlands). Nme was grown onGC agar (Becton Dickinson) plates containing Vitox (Oxoid) andantibiotics when appropriate (kanamycin 100 μg/ml, chloramphenicol 5μg/mi) in candle jars at 37° C. Liquid cultures were grown in trypticsoy broth (TSB) in plastic flasks at 37° C. with shaking. Forsialylation experiments, 80 μM cytidine 5′ monophospho-N-acetylneuraminic acid (CMP-NANA, Sigma) was added for 2 h to the medium ofbacteria growing in mid-log phase. E. coli strains DH5 α or TOP10F′(Invitrogen) were used for routine cloning. E. coli was propagated on LBplates. Antibiotics were added in the following concentrations:kanamycin 50 μg/ml, chloramphenicol 25 μg/ml and erythromycin 200 μg/ml.

Gel Electrophoresis and Immunoblotting.

SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing orsemi-native conditions and immunoblotting were performed as described(Voulhoux et al 2003 Science 299; 262-265). For LPS evaluation, sampleswere boiled in SDS-PAGE sample buffer and subsequently incubated with0.5 mg/ml proteinase K at 55° C. for one hour. After boiling for 10 min,lysates were run on 16% Tricine-SDS-PAGE (Lesse et al, 1990, J. Immunol.Methods. 126; 109-117) and stained with silver (Tsai et al 1982, Anal.Biochem. 119; 115-119).

Neuraminidase Treatment.

One ml of bacteria grown to mid-log phase was pelleted and washed withbuffer A (20 mM Na2HPO4/NaH2PO4, 150 mM NaCl, 5 mM MgCl2, 5 mM CaCl2, pH6.0). Bacteria were resuspended in 0.5 ml buffer A and 0.2 U/mlneuraminidase (type V, Clostridium perfringens, Sigma N-2876) was addedfor 60 min at 37° C. Next, bacteria were pelleted and processed forTricine-SDS-PAGE. Cell envelopes were diluted in buffer A and incubatedwith 0.2 U/ml neuraminidase for 60 min at 37° C.

Isolation of Cellular Fractions.

Cell envelopes were prepared as described (Voulhoux et al 2003 Science299; 262-265). Inner and outer membranes were separated by isopycnicsucrose-gradient centrifugation according to Masson and Holbein (Massonand Holbein 1983, J. Bacteriol. 154; 728-736) or, alternatively,according to the procedure of Shell et al. (Shell et al 2002, Infect.Immun. 70; 3744-3751). Lactate dehydrogenase activity was measureddirectly in the sucrose-gradient fractions (Westphal and Jann 1965;Method. Carbohydr. Chem. 5; 83-91). Equal volumes of each fraction wereprecipitated with 7% trichloroacetic acid (TCA) and analysed forproteins by SDS-PAGE and for LPS by Tricine-SDS-PAGE. To obtainextracellular growth medium, bacteria were removed from suspensions bycentrifugation (15 min 6000 g). The supernatant was spun for 2 h at100.000 g. Proteins and LPS were precipitated from the supernatants with7% TCA. The precipitates were collected by centrifugation at 20.000 gfor 30 min followed by an acetone wash.

LPS Quantification.

The LPS content of cell envelopes was determined by3-deoxy-D-manno-octulosonic acid (KDO) measurement as described (VanAlphen et al 1978; J. Bacteriol. 134; 1089-1098).

Antibodies.

Overexpression of the Imp protein in the cell envelope of H44/76 wasachieved by growing the imp mutant carrying the plasmid pEN11-Imp with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG). These induced cells wereused to prepare outer membrane vesicles (Fredriksen et al 1991, NIPHAnnals 14, 67-79) that were injected into mice to raise antiserum. Next,specific anti-imp antibodies were purified by adsorption of the sera topurified Imp protein. For that end, inclusion bodies from strain BL21pET11 a-Imp were purified (Dekker et al 1995; Eur. J. Biochem. 232;214-219), dissolved in 20 mM Tris/HCl, 100 mM glycine, 6 M urea pH 8,electrophoresed in 8% SDS-PAGE gels and blotted onto nitrocellulose. TheImp protein was visualized on the blot using 0.25% Ponceau S (AcrosOrganics) in 1% acetic acid. A strip containing the Imp protein was cutfrom the blot and used to adsorb specific anti-Imp antibodies from thesera of the immunized mice. Bound antibodies were eluted by a 5 min washwith 0.2 M glycine pH 3.0 followed by neutralization with 1 M Tris pH10.8. The eluted antibodies were used for the specific detection of Impon blots. Mouse monoclonal anti-FbpA and anti-PorA (MN23G2.38)antibodies were gifts from B. Kuipers (NVI, Bilthoven, The Netherlands).

Analysis of PL Composition

Cells grown overnight on plate were harvested and resuspended in TSB.After subsequent dilution in 5 ml TSB to an OD550 of 0.1, cells werelabeled for 7 h with 2 μCi [1-14C] sodium acetate at 37° C.Phospholipids were isolated from 1.4 ml of culture (Bligh and Dyer, 1959Can. J. Med. Sci 37; 911-917), separated by TLC, and plates (silica gel60, 20×10 cm, Merck) were developed with chloroform/methanol/acetic acidat a ratio of 65:25:10 and subjected to autoradiography.

LPS and Phospholipid Isolation and Quantification

SDS-PAGE under denaturing conditions was performed as described(Voulhoux et al., 2003 Science 299; 262-265). For LPS analysis, sampleswere boiled in SDS-PAGE sample buffer and subsequently incubated with0.5 mg/ml proteinase K at 55° C. for one hour. After boiling for 10 min,lysates were analyzed on 16% Tricine-SDS-PAGE (Lesse et al., 1990 J.Immunol. Methods 126; 109-117) and stained with silver (Tsai and Frasch,1982 Anal. Biochem. 119; 115-119). Cell envelopes were isolated asdescribed previously (Voulhoux et al, 2003 Science 299; 262-265). TheLPS content of cell envelopes was determined by KDO measurement asdescribed (van Alphen et al., 1978 J. Bacteriol. 134; 1089-1098). Cellswere harvested from plate and washed with a buffer containing 0.238%free acid HEPES, 0.04% KCl, 0.85% NaCl, 0.01% MgCl2.6H20, 0.09%anhydrous glucose, and 0.5 mM CaCl2, adjusted with NaOH to pH 7.4.Phospholipids were isolated as described (Bligh and Dyer, 1959 Can. J.Med. Sci 37; 911-917) and the amount was quantified by determining thephosphorus content (Rouser et al., 1970 Lipids 5; 494-496).

Electron Microscopy

Cells were harvested from plate and chemically fixed, embedded ingelatin and cryosectioned. Ultrathin sections were observed with aTechnai 10 EM at 100kV.

EXAMPLE 2 Imp is Not Essential in N. Meningitidis.

A Neisserial imp mutant was constructed by allelic replacement of theimp gene in strain H44/76 with a copy containing a deletion-insertionmutation (FIG. 1A). We used the sequence of NMB0279 and NMB0280 fromstrain MC58 (http://www.tigr.org) to design primers to clone andsubsequently delete the imp gene in Nme strain H44/76 (FIG. 1A).Briefly, part of the gene upstream of imp, NMB0279, was cloned fromH44/76 DNA using primers A and B (Table 1). The 3′ end of the imp genewas obtained by PCR with primers C and D. Both PCR products were clonedinto pCR2.1-TOPO (Invitrogen), resulting in plasmids pCR2.1- NMB0279 andpCR2.1-3′ imp. An AccI-XbaI fragment of pCR2.1-NMB0279 was ligated intoAccI-XbaI restricted pCR2.1-3′imp. The resulting plasmid was cut withAccI to allow insertion of a kanamycin-resistance cassette. Thiscassette was PCR amplified from plasmid pACYC177 (New England Biolabs)using primers E and F (Table 1), which introduced terminal AccI sitesand a Neisserial DNA uptake sequence. The final construct, called pMB25,contained the kanamycin-resistance cassette in the same orientation asthe transcription direction of the imp gene. Approximately 200 ng of apurified PCR product amplified from pMB25 with primers A and D was addedto wild-type H44/76 bacteria growing in TSB plus 10 mM MgCl2 for 6 h.Bacteria were plated on GC plates containing kanamycin. Transformantswere screened by PCR using primer pairs AD, AF and DE. Forcomplementation experiments, we cloned the imp gene from H44/76 genomicDNA by PCR using the primer pair D and G (Table 1).

Table 1. Oligonucleotides (primers) used in this study. Underlinedsequences indicate restriction sites: AccI in primers B, C, E and F;NdeI in primers G and H, AatII in primer D and BamHI in primer I. Dashedline in primer F indicates the Neisserial DNA uptake sequence.

Sequence (5′-3′) Purpose A ATGCCTGCAACCTTCAAGTG, 5′ primer for SEQ IDNO: 18 cloning of NMB0279 B ATGTCGACAATCGCCCCTCAAGTCGGTTTG, 3′ primerfor SEQ ID NO: 19 cloning of NMB0279 C ATGTCGACTACCTGCGGCCGGATTATGC,5′ primer for SEQ ID NO: 20 cloning of 3′ end of imp DATGACGTCTCAGGGTCGTTTGTTGCGTCC 3′ primer for GGC, cloning of 3′ SEQ IDNO: 21 end of imp E AGCGTCGACTTCAGACGGCCACGTTGTGTC, 5′ primer for SEQ IDNO: 22 cloning of Kan-cassette F AGCGTCGACGCTGAGGTCTGCCTCGTG, 3′ primerfor SEQ ID NO: 23 cloning of Kan-cassette GATCATATGGCTCGTTTATTTTCACTCAAACC, 5′ primer for SEQ ID NO: 24 cloning ofcom- plete imp gene into pEN11 H TGCATATGGATGCCGTTGCGGCGGAG, 5′ primerfor SEQ ID NO: 25 clongin of imp into pET11a ITGGGATCCTCAGGGTCGTTTGTTGCGTCC, 3′ primer for SEQ ID NO: 26 cloning ofimp into pET11a

The PCR product was cloned in pCR2.1-TOPO, cut and ligated into pEN11using NdeI and AatiI restriction, resulting in plasmid pEN11-Imp.Plasmid pEN11, a Neisseria-replicative plasmid, is a derivative ofRV2100, which contains the H44/76 omp85 gene behind a tandem lacpromoter-operator (tac-lacUV5) sequence (Voulhoux et al 2003 Science299; 262-265). In pEN11, the ATG initiation codon of the omp85 gene isreplaced by an NdeI site to facilitate exchange of genes. The imp mutantwas transformed with pEN11-Imp by coincubation of bacteria with plasmidfor 6 h on plate (Voulhoux et al 2003 Science 299; 262-265).Transformants were selected on plates containing chloramphenicol andtested for the presence of pEN11-Imp and the chromosomal imp::kan alleleby PCR. The H44/76 imp gene without its signal sequence was cloned inpET11a (Novagen) using primers H and I (Table 1). The resulting plasmidpET11a-Imp was introduced into E. coli strain BL21 (DE3) (Novagen) toallow expression of the truncated imp gene from the T7 promoter presentin pET11a.

Kanamycin-resistant transformants were tested by PCR for the absence ofan intact copy of the imp gene and the presence of the imp::kan allele.Correct transformants were readily obtained, demonstrating that incontrast to E. coli (Braun & Silhavy 2002, Mol. Microbiol. 45;1289-1302), imp is not an essential gene in Nme. The absence of the Impprotein in the mutants was confirmed by immunoblotting (FIG. 1B).

EXAMPLE 3 Phenotype of a Neisserial Imp Mutant.

A striking feature of the transformants was their intense colony opacitycompared to wild-type colonies (FIG. 2A, B), a property also apparentfor the LPS-deficient mutant (FIG. 2C). Furthermore, similar to theLPS-deficient strain (Steeghs et al 2001; EMBO J. 24; 6937-6945), theimp mutant bacteria grew slower and to a lower final optical densitythan wild-type bacteria (FIG. 2D). Analysis of the protein profiles ofwhole cell lysates (data not shown) or cell envelopes (FIG. 3A) indenaturing or semi-native SDS-PAGE showed no marked differences betweenwild-type and imp mutant bacteria. The major OMPs of Nme are thetrimeric porins PorA and PorB. These trimers are very stable and do notdissociate into monomers during semi-native SDS-PAGE (Voulhoux et al2003 Science 299; 262-265). When we analyzed cell envelopes of the impmutant in semi-native conditions, most of the PorA protein was presentin its trimeric form, as shown by immunoblotting (FIG. 3B). Only a smallamount of monomeric porA was detected in the imp mutant analogous to theprofile of the IpxA mutant (FIG. 3B) (Steeghs et al 2001; EMBO J. 24;6937-6945). Thus, OMPs such as PorA and PorB are present in normallevels and are assembled correctly. In contrast, Tricine-SDS-PAGEanalysis showed that the cellular LPS content was dramatically decreasedin the imp mutant (FIG. 3C). Quantitative measurements of LPS, bydetermining the levels of KDO, an intrinsic component of the coreregion, confirmed this result: the imp mutant cell envelopes containedonly 6.4 nmol KDO/mg protein, whereas wild-type levels were 95 nmolKDO/mg protein. The LPS of the imp mutant migrated at a similar positionin the gel as wild-type LPS (FIG. 3C), indicative of similar sizes. Thepossibility that LPS was released by the imp mutant bacteria wasinvestigated by analyzing extracellular growth media on TricineSDS-PAGE. No enhanced release of LPS by the imp mutant bacteria wasfound (data not shown). In contrast, the wild-type and imp mutant showedvery different extracellular protein profiles (FIG. 3D). The majorprotein present in the medium of the imp mutant was an approximately35-kDa protein, which could be identified by immunoblotting as FbpA(data not shown), a periplasmic iron transporter (Ferreiros et al 1999.Comp. Biochem. Physiol. 123; 1-7). Similar high levels of FbpA werefound in the extracellular medium of the IpxA mutant (FIG. 3D). Theseresults indicate periplasmic leakage occurring in the imp and IpxAmutants, a phenomenon also reported for E. coli mutants expressingreduced amount of LPS (Nurminen et al 1997′ Microbiology 143;1533-1537). Complementation of the imp mutation by introduction of theimp gene on a plasmid under the control of an IPTG-regulatable promoterinto the imp mutant resulted in complete restoration of all wild-typephenotypic traits described above in the presence of IPTG (data notshown), demonstrating that the imp mutant phenotype is directly relatedto Imp deficiency. Thus, the imp mutant demonstrates a similar phenotypeas the IpxA mutant, indicative of a role of Imp in LPS biogenesis. Incontrast to the IpxA mutant however, the imp mutant still produced a lowamount of apparently full-length LPS. The presence of intact LPSmolecules argues against a defect in LPS biosynthesis in the imp mutant.The low levels of LPS found may rather result from feedback inhibitionon LPS synthesis by mislocalized LPS.

EXAMPLE 4 Localisation of LPS in Imp Mutant Strains by MembraneSeparation.

In order to localize the LPS produced by the imp mutant, we performedsucrose-gradient density centrifugation to separate inner and outermembranes. Despite many attempts using different protocols, we neverobtained satisfactory membrane separations even of wild-type cells. Asexpected, the inner membrane marker, lactate dehydrogenase, fractionatedto the lighter density fractions (FIG. 4A), whereas the OM porinsfractionated mostly to the heavier fractions (FIG. 4B). However, LPS wasfound in almost every fraction of the gradient (FIG. 4C) and did notco-fractionate with the porins. Difficulties with Neisserial membraneseparations were also appreciated previously (Masson & Holbein 1983, J.Bioteriol. 154; 728-736). The LPS of the imp mutant fractionatedsimilarly in sucrose gradients as the LPS of the wild-type strain (datanot shown), but because of the non-conclusive results with the wild-typemembranes, we would not want to draw any conclusion from these results.Instead, we designed an alternative method to assess LPS localization inthe imp mutant.

EXAMPLE 5 Surface Accessibility of LPS.

Neisseriae do not synthesize O-antigen. The terminal oligosaccharideportion of the core of Neisserial LPS is variable due to phase-variableexpression of the glycosyltransferases involved. Consequently, manydifferent so-called LPS immunotypes exist. The L3 immunotype contains alacto-N-neotetraose unit as terminal oligosaccharide of the α-chain,which can be further extended by a sialic acid residue. Meningococci arecapable of sialylating the lacto-N-neotetraose unit by usingendogeneously produced CMP-NANA as substrate donor or by utilizing thisnucleotide sugar when added to the growth medium (Kahler & Stephens1998, Crit. Rev. Microbiol. 24; 281-334). The sialic acid residue can beremoved from LPS by treating intact bacteria with neuraminidase (Ram etal 1998, J. Exp. Med. 187; 743-752). We utilized this feature to assessthe cell surface location of LPS. The results described so far wereobtained with an Nme L8 immunotype that cannot be sialylated. To exploitthe neuraminidase assay, we constructed an imp mutant in an L3background. The phenotype of this mutant, in terms of colony opacity,growth characteristics, release of periplasmic protein (data not shown)and low LPS content (FIG. 5A), was identical to that of the L8 impmutant. The LPS of the L3 imp mutant appeared in silver-stainedTricine-SDS-PAGE gels as two bands (FIG. 5A, B). After neuraminidasetreatment of cell envelopes, all LPS migrated at the lower position(FIG. 5B), demonstrating that the higher band corresponds to sialylatedLPS. After growth of the mutant in the presence of CMP-NANA, all LPSmigrated at the higher position, and was completely converted to thelower migrating form upon neuraminidase treatment of cell envelopes(FIG. 5B). Thus, the L3 imp mutant produces LPS with a full-length a-chain which can be completely sialylated and subsequently bedesialylated with neuraminidase. Wild-type bacteria produced sialylatedLPS only when CMP-NANA was added to the growth medium (FIG. 5B);apparently the endogeneous CMP-NANA levels are rate-limiting whenregular high levels of LPS are produced.

To test whether LPS was exposed at the cell surface, we treated intactbacteria grown in the presence of CMP-NANA with neuraminidase. Only aminor part of LPS was desialylated in the intact imp mutant cells,indicating that most of the LPS was not accessible to neuraminidase atthe cell surface (FIG. 5C). The small amount of LPS that was accessible,possibly resulted from the leakiness of the mutant cells, as revealed bythe enhanced protein release observed (FIG. 3D). In contrast, sialylatedLPS present in wild-type cells was completely desialylated and thusfully exposed at the cell surface as expected (FIG. 5C). To addresswhether the difference in neuraminidase accessibility between wild-typeand imp mutant bacteria was influenced in any way by the largedifference in total LPS present, we performed similar assays in a strainwhere IpxA expression is regulatable with IPTG (Steeghs et al 2001; EMBOJ. 24; 6937-6945). This strain was grown in the presence of CMP-NANA andvarious concentrations of IPTG. Expression of LPS was dependent on theIPTG concentration used, although we detected some LPS even in theabsence of IPTG (FIG. 5D); apparently the IPTG-inducible promoter wasnot completely silent. Nevertheless, at all different cellular LPSlevels, cell surface localization of LPS was evident as inferred fromits full accessibility to neuraminidase in intact cells (FIG. 5D). Thesedata further validate the assay used and therefore strengthen ourconclusion that LPS is mostly absent from the cell surface in the impmutant. Thus, Imp functions in LPS transport to the outer leaflet of theOM.

EXAMPLE 6 Imp Homologs in Other Bacteria.

The sequence of the Nme MC58 imp gene NMB0280 (http://www.tigr.org) wasused as a query to search microbial genomes for Imp homologues usingBLAST. Molecules involved in the biogenesis of well-conserved structuressuch as LPS are likely highly conserved. This is indeed the case for theimp gene, since homologs can be found in most Gram-negative, but not inGram-positive bacteria (Braun & Silhavy 2002, Mol. Microbiol. 45;1289-1302). The absence of an imp homolog in some Gram-negative bacteriaappears to correlate with the absence of LPS, since we were unable tofind imp homologs in bacteria that posses an outer membrane, but lackLPS biosynthesis genes (Raetz et al 2002, Annu. Rev. Biochem. 71;635-700), such as Thermotoga maritima, Deinococcus radiodurans and thespirochaetes Borrelia burgdorfferi and Treponema pallidum. Thisobservation further reinforces the notion of Imp functioning as an LPStransporter.

EXAMPLE 7 Topology model of Imp

In order to understand the mechanism of Imp-mediated LPS transport, atopology model was made of Neisserial Imp. Our topology model predicts18 transmembrane beta strands. With short periplasmic turns and somevery long (60 amino acid residues) extracellular loops (FIG. 6A). Thelong loops are quite remarkable since they are very well conserved amongNeisserial Imp proteins (FIG. 7).

Discussion

LPS is an essential component of the outer membrane of mostGram-negative bacteria and a causative agent of severe septic shock inhumans. Its biogenesis has been studied for a long time, resulting inthe identification of many proteins involved in its biosynthesis.However, the final step of LPS biogenesis, i.e. the transport ofcompleted LPS molecules from the periplasmic leaflet of the IM to thebacterial cell surface has remained elusive. We have now identified forthe first time a protein required for this LPS transport pathway. ANeisserial imp mutant produced drastically reduced amounts offull-length LPS. Although we were unable to determine exactly thecellular location of the limited amount of LPS that accumulated in theimp mutant, the neuraminidase accessibility assay clearly showed thatthe vast majority of this LPS was not accessible at the cell surface.Since Imp itself is an OMP, as shown by its presence in purified E. coliouter membranes and indicated by its high content of aromatic residues(Braun & Silhavy 2002, Mol. Microbiol. 45; 1289-1302) typical ofβ-barrel OMPs, Imp is likely the transporter that mediates theflip-flopping of LPS over the OM, although an additional role of Imp intransport through the periplasm cannot be excluded at this stage. Thestrongly decreased amounts of LPS in the imp mutant might be due tofeed-back inhibition of LPS biosynthesis by mislocalized LPS.

Braun and Silhavy (Braun & Silhavy 2002, Mol. Microbiol. 45; 1289-1302)reported that depletion of Imp in a conditional E. coli mutant resultedin the appearance of novel, high-density membranes found in sucrosegradient fractionations. This higher density might result from anincreased protein to lipid ratio. Consistently, whereas OMP assemblyappeared unaffected by Imp depletion, both in E. coli (Braun & Silhavy2002, Mol. Microbiol. 45; 1289-1302) and in Nme (this study), wedemonstrated now that Imp depletion results in decreased levels of LPSin the OM, thus changing the protein:lipid ratio. Also, the observationsthat missense mutations in the E. coli imp gene resulted in increasedsensitivity to hydrophobic agents (Sampson et al 1989 Genetics 122;491-501: Alono et al 1994, Appl. Environ. Microbiol. 60; 4624-4626) cannow be understood: these mutants likely suffered from reduced levels ofLPS, a property known to affect the integrity of the OM (Nurminen et al1997, Microbiology 143; 1533-1537).

Previously, another essential OMP, Omp85, has been suggested to beinvolved in LPS transport (Nurminen et al 1997, Microbiology 143;1533-1537). However, we have demonstrated a strong OMP assembly defectin an Omp85-depleted strain (Voulhoux et al 2003 Science 299; 262-265).Thus, any effect of Omp85 depletion on LPS biogenesis might be aconsequence of the misassembly of Imp. Furthermore, the demonstration ofan interaction of Omp85 with non-native porin (Voulhoux et al 2003Science 299; 262-265), the presence of an omp85 homolog in Gram-negativebacteria lacking LPS biosynthesis genes and the high conservation of Impin Gram-negative bacteria, except in those that lack LPS-biosynthesisgenes (this study), all argue for a direct role of Omp85 in OMP assemblyand of Imp in LPS transport. With the identification of the functions ofOmp85 and Imp, major progress in understanding the biogenesis of thebacterial outer membrane can now be made.

The Imp protein is an attractive target for the development of novelantibacterial substances, in light of its high conservation, cellsurface localization and essential role in most Gram-negatives.Additionally, Neisserial imp mutant strains might be useful as vaccinestrains. Neisserial vaccines consist of outer membrane vesicles that aretreated with detergents to remove the majority of LPS in order toprevent toxic reactions in vaccinees. This procedure unfortunatelyremoves also potentially important vaccine components such ascell-surface exposed lipoproteins. Vaccines prepared in this way containapproximately 7% of normal LPS levels (Fredriksen et al 1991, NIPHAnnals 14, 67-79). Our data show that that is about the level of LPSleft in the imp mutant. Thus, deletion of the imp gene in a vaccinestrain relieves the need for detergent extraction and thereby the lossof potentially important vaccine components.

The Imp protein was named after the phenotype of the imp missensemutants (increased membrane permeability). We propose to change thisname now that we have established the function of Imp. We suggest toname the gene IpxZ, in line with the Ipx designation used for LPSbiogenesis genes and the Z to signify that the imp gene product mediatesthe final step in LPS biogenesis.

EXAMPLE 8 Construction of Plasmids and msbA-mutant Strains

To disrupt the msbA gene in N. meningitidis, we made use of theavailable genome sequence of strain MC58 (Tettelin et al, 2000 Science287; 1809-1815) to design PCR primers (FIG. 9). Briefly, parts of thegenes upstream and downstream of msbA, designated NMB1918 and NMB1920,respectively, were amplified by PCR from genomic DNA of H44/76 using Taqpolymerase and primer pairs A/B and C/D, respectively (FIG. 9). Both PCRproducts were cloned into pCRII-TOPO (Invitrogen), resulting in plasmidspCRIINMB1918 and pCRII-NMB1920, respectively. An AccI-KpnI fragment ofpCRIINMB1918 was ligated into AccI-KpnI digested pCRII-NMB1920. Theresulting plasmid was cut with AccI to allow for the insertion of akanamycin-resistance cassette derived from pMB25 (Bos et al, 2004 Proc.Natl. Acad. Sci. USA). The final construct, called pBTmsbA::kan,contained the kanamycin-resistance cassette in the same orientation asoriginally the msbA gene and was used as the template for amplificationof the disruption fragment by PCR with primer pair A/D (FIG. 9).Approximately 200 ng of this PCR product was added together with 5 mMMgCl2 to H44/76 or HB-1 bacteria that were subsequently grown on platefor 6 h. Hereafter; bacteria were transferred to plates containingkanamycin. The correct gene replacement in kanamycin-resistanttransformants was confirmed by PCR using primer pair A/D.

For complementation experiments, we cloned the msbA gene from H44/76genomic DNA by PCR with primer pair E/F (FIG. 9) using the High FidelityKit (Roche) according to manufacturer's protocol. The PCR product wascloned into pCRII-TOPO, ligated into pEN11 (Bos et al., 2004) after NdeIand AatII restriction, resulting in plasmid pEN11-msbA. The msbA mutantderived from strain H44/76 was transformed with pEN11 -msbA bycoincubation of bacteria with plasmid and 5 mM MgCl2 for 6 h on plate.Transformants were selected on plates containing chloramphenicol andrepeatedly restreaked on plates containing 100 μMisopropyl-β-D-thiogalactopyranoside before performing complementationexperiments. All enzymes were provided by Fermentas, except whereindicated otherwise.

EXAMPLE 9 MsbA is not Essential for N. meningitidis

The genomes of N. meningitidis strains MC58 (Tettelin et al., 2000) andZ2491 (Parkhill et al., 2000 Nature 404; 502-506) were searched with thedefault search matrix of the tBlastn program (Altschul et a/., 1997Nucleic Acids Res. 25; 3389-3402) using the amino acid sequence of E.coli MsbA as a probe (http://www.ncbi.nlm.nih.gov/blast). The amino acidsequence of the putative MsbA protein encoded by the MC58 gene NMB1919displayed 32% identity and 52% similarity to that of E. coli MsbA. Asimilar degree of homology (31% identity and 52% similarity,respectively) was found for the putative MsbA protein of Z2491. An msbAmutant was constructed by allelic replacement in N. meningitidis strainH44/76 (FIG. 9). Kanamycin-resistant transformants were analyzed by PCRto verify the absence of an intact copy of the msbA gene and thepresence of the msbA::kan allele. Since correct transformants wereobtained at high frequency, it appears that in N. meningitidis, incontrast to E. coli (Zhou et al, 1998 J. Biol. Chem. 273; 12466-12475),MsbA is not essential for viability.

EXAMPLE 10 LPS Content of the msbA Mutant

Proteinase K-treated cell lysates from approximately 2.107 cells (basedupon the estimation that an optical density at 550 nm (OD550) of 1represents 1.109 cells/ml) from both wild-type and msbA-mutant cellswere analyzed by Tricine-SDS-PAGE (FIG. 10A). Whereas LPS could clearlybe detected on the gels in the cell lysate from the wild-type strain, itwas barely visible in the cell lysate of the msbA mutant strain (FIG.10A). Apparently, the msbA mutation has a strong impact on LPSsynthesis, possibly due to some feedback inhibition mechanism caused byLPS stalled in the transport pathway, as previously observed in the impmutant (Bos et al., 2004 Proc. Natl. Acad. Sci. USA). To quantify theLPS content, we determined the amount of 3-deoxy-D-mannooctulosonic acid(KDO), a structural component typical for LPS, in wild-type and mutantcells. Cell envelopes of the msbA mutant cells contained an LPS toprotein ratio of 7% when compared to wild-type cells and similar to thatin the imp mutant (FIG. 10B). Since a putative transcriptionalterminator is present immediately downstream of the msbA gene (FIG. 9),the decreased LPS content in the msbA mutant was expected to be a directconsequence of the inactivation of the msbA gene and not of any polareffects of the mutation on downstream located genes. This suppositionwas confirmed in a complementation experiment. When plasmid pEN11-msbA,carrying a wild-type msbA gene, was introduced into the msbA mutant, theLPS to protein ratio was restored to nearly wild-type levels (FIG. 10B).

EXAMPLE 11 Growth Characteristics

As described previously for the IpxA mutant (Steeghs et al., 1998 Nature392; 449-450) and the imp mutant (Bos et al, 2004 Proc. Natl. Acad. Sci.USA), the generation time of the msbA null mutant was strongly reducedduring exponential growth as compared to the wild type and the culturesdid not reach the same final OD as those of the wild-type strain (FIG.11). Additionally, after 16 h growth at 37° C. the colonies of the msbAmutant, like those of the IpxA and imp mutants (Bos et al., 2004 Proc.Natl. Acad. Sci. USA), were smaller than those of the wild type and theyalso had an opaque appearance, in contrast to those formed by thewild-type strain (data not shown). Interestingly, the colonies of themsbA mutant were heterogeneous, with either smooth-edged orlobated-edged colonies (data not shown). The ratio of these two types ofcolonies seemed to increase from ˜1 to ˜20 in favor of the latter whensamples were taken at different points during exponential growth (datanot shown). N. meningitidis cells grown in liquid culture undergoautolysis, several hours after entering the stationary growth phase asshown in FIG. 11. This is described as being a result of the activity ofthe OM phospholipase A (OMPLA) (submitted OMPLA paper M. P. Bos). In thecase of the msbA mutant, autolysis was retarded (FIG. 11). The cells dideventually lyze, but only after prolonged incubation periods (data notshown), a phenotype which was also observed for the imp mutant(unpublished results). Possibly, OMPLA requires LPS for activity, as hasbeen described previously for another OM enzyme, i.e. the protease OmpTof E. coli (Kramer et al., 2002 Eur. J. Biochem. 269; 1746-1752).

EXAMPLE 12 Electron Microscopy and Cell Envelope Protein Profile

To determine whether the msbA mutant cells still have a double membrane,we prepared ultrathin sections and examined them by electron microscopy(FIG. 12A,B). Indeed, a double membrane was clearly visible indicatingthat both IM and OM were still present. Apparently, the msbA mutationdid not prevent the formation of an outer membrane. Additionally,analysis of the cell envelope protein profiles indicated that theexpression of the major OM proteins PorA and PorB is not compromised inthe msbA mutant (FIG. 12C). These results are comparable to thoseobtained with the IpxA (Steeghs et al, 1998 Nature 392; 449-450) and imp(Bos et al, 2004 Proc. Natl. Acad. Sci. USA) mutants. In conclusion, itappears that the msbA mutant is still able to assemble an OM, suggestingthat PL transport is not compromised in the msbA mutant.

EXAMPLE 13 Phospholipid Composition of the msbA Mutant

To investigate whether all major PL species were produced in the msbAmutant, cells were labeled with [14C] sodium acetate, and PL wereextracted and analyzed by thin layer chromatography (TLC) (FIG. 13A). N.meningitidis was previously reported to produce large amounts ofphosphatidylethanolamine (PE) and phosphatidylglycerol (PG), minoramounts of phosphatidic acid (PA) and trace amounts of cardiolipin (CL)(Rahman et a/., 2000 Microbiology 146; 1901-1911). When the PL profileof the msbA mutant was compared with that of the wild-type strain, nodrastic change in PE content was observed (FIG. 13A). However, theamount of PG relative to that of PA and CL, which run at the sameposition in the TLC system used here, seemed clearly decreased (FIG.13A). The same characteristics were found for the imp mutant (data notshown). The lack of LPS in the OM of LPS biogenesis mutants must becompensated by other lipidic components to form an OM. To investigatewhether the msbA mutant produced more PL than did wild-type cells, PLwere extracted from cells grown on plate and quantified by phosphorusdetermination. The msbA mutant derived from wild-type strain H44/76,which possesses a capsule, showed no increase in the total amount of PL(data not shown). Strikingly, however, the msbA mutant of strain HB-1,which produces no capsule, showed a considerable (p<0.06) increase inthe total amount of PL compared to its parental strain (FIG. 13B).Apparently, in this strain, increased PL levels compensate the lack ofLPS, whereas in the msbA mutant of strain H44/76 the lack of LPS mightby compensated by increased amounts of capsule, which is anchored viaits lipid tail in the outer leaflet of the outer membrane.

EXAMPLE 14 Complementation of a Temperature-sensitive msbA Mutant of E.coli

The results presented so far suggest that in N. meningitidis MsbA isrequired only for LPS transport, whereas in E. coli, MsbA has beenreported to be required for transport of both LPS and PL (Zhou et al.,1998 J. Biol. Chem. 273; 12466-12475). This discrepancy could beexplained by assuming that the two MsbA proteins have overlapping, butdifferent functions. To test this possibility, we investigated whetherN. meningitidis msbA can complement an E. coli msbA mutation. The growthof the E. coli K-12 temperature-sensitive msbA strain WD2 is arrested at44° C. (Doerrier et al, 2001 J. Biol. Chem. 276; 11461-11464). WhenpEN11-msbA, containing the msbA gene of N. meningitidis, was introducedinto WD2, growth was fully restored at 44° C. to wild-type levels (datanot shown). Apparently, the Neisserial MsbA protein can functionallycomplement the E. coli MsbA.

Discussion

Based on the analysis of a temperature-sensitive msbA mutant of E. coliMsbA has been suggested to be involved in both LPS and PL transport(Zhou et a/., 1998 J. Biol. Chem. 273; 12466-12475). However, recent invitro analysis indicated that, in contrast to several other integral IMproteins, MsbA reconstituted in proteoliposomes did not stimulate PLflip-flop (Kol et al., 2003 J. Biol. Chem. 278; 24586-24593). It waspostulated that a subset of proteins, characterized by a small number oftransmembrane helices, facilitate lipid translocation via theprotein-lipid interface (Kol et al/, 2004 Biochemistry 43; 2673-2681).These proteins could be involved in this process, because they displaymore dynamic behavior and engage in less stable protein-lipidinteractions than larger membrane proteins (Kol et al, 2004 Biochemistry43; 2673-2681). However, it remained a possibility that MsbA is requiredfor the release of PL from the outer leaflet of the IM for subsequenttransport through the periplasm to the OM. To investigate whether MsbAhas a role in PL transport, we made use of the ability of N.meningitidis to survive without LPS. The expectation was that it wouldbe impossible to generate an msbA mutant if the MsbA protein had anessential role in the transport of PL, whereas the gene would bedispensable if its product were involved in LPS transport only. We foundthat an msbA disruption mutant could be created, thereby excluding anessential role for MsbA in PL transport. The mutant showed drasticallyreduced LPS levels, consistent with a role for MsbA in LPS biogenesis.The reduced levels of LPS in the msbA mutant might be the result offeedback regulation on LPS synthesis by LPS molecules stalled in thetransport pathway, similarly as previously reported for the imp mutant(Bos et al., 2004 Proc, Natl. Acad. Sci. USA). Although the growth ratewas clearly affected by the msbA mutation, an OM was still present andthe major OM protein profile was similar to that of the wild type. Allthe major PL were produced in the msbA mutant, although the amount of PGseemed somewhat decreased, whereas the total amount of PA and CL seemedsomewhat increased. The change in the PL profile could be a response tothe loss of LPS from the OM, as the imp mutant showed the same phenotypein this respect. In addition, in the msbA mutant derived from HB-1,which lacks a capsule, PL were overproduced in such amounts, that theycould form the outer leaflet in the OM, thereby replacing LPS.Similarly, it has been shown previously in E. coli that mutations in theLPS biosynthesis genes, htrB (IpxL) (Karow et al., 1992 J. Bacteriol.174; 7407-7418) and IpxC (Kloser et al., 1998 Mol. Microbiol. 27;1003-1008) gave rise to higher PL levels. However, such an increase inPL content was not observed in the msbA mutant of the capsule-producingstrain H44/76. Previously, the impossibility to create an IpxA mutationin a N. meningitidis strain lacking capsule was reported (Steeghs et al,2001 EMBO J. 20; 6937-6945). Possibly, the small amount of LPS stillmade in the msbA mutant allowed for the construction of an msbA mutantin this background, even if these LPS molecules were not correctlylocalized. Importantly, a low-copy vector containing the msbA gene of N.meningitidis could complement a temperature-sensitive msbA mutant of E.coli Since N. meningitidis MsbA is involved in LPS transport only, thisresult suggests that MsbA of E. coli is not required for PL transporteither. The accumulation of PL in the IM observed in such an E. colimutant at the restrictive temperature (Doerrier et al., 2001 J. Biol.Chem. 276; 11461-11464) could then be explained as a secondary effect ofthe defective LPS transport.

1. An outer membrane vesicle preparation from a Neisserial bacteriumthat expresses Imp protein wherein expression of the Imp protein isfunctionally downregulated by disrupting the structure of the Impprotein such that the level of lipopolysaccharide in the outer membraneis decreased compared to a wild-type Neisserial bacterium, wherein atleast one of the extracellular loops of the Imp protein is disrupted byinserting a sequence from a different protein into the loop to make achimeric protein, wherein the chimeric protein comprises at least onepart from an Imp protein and at least one part from a Neisserial Hsfprotein, wherein the part from a Neisserial Hsf protein is inserted intoloop 8 of the Imp protein.
 2. The outer membrane vesicle preparation ofclaim 1 derived from N. meningitidis wherein the amount of LPS in theouter membrane vesicle is reduced compared to the amount of LPS in anouter membrane vesicle preparation derived from a strain of N.meningitidis where Imp or MsbA is not functionally disrupted.
 3. Theouter membrane vesicle preparation of claim 1 wherein the level of LPSis sufficiently low so that the toxicity is reduced to a level at whichthe outer membrane vesicle preparation has an acceptable level ofreactogenicity when inoculated into a patient.
 4. The outer membranevesicle preparation of claim 1 wherein LPS present in the outer membranevesicles is intra-vesicle cross-linked to outer membrane proteins in theouter membrane vesicle.
 5. The outer membrane vesicle preparation ofclaim 1 wherein the concentration of lipoproteins in the outer membranevesicles is equivalent to the concentration of lipoproteins from outermembrane vesicles derived from a non-detergent extraction process.
 6. Apharmaceutical composition comprising the outer membrane vesiclepreparation of claim 1 and a pharmaceutically acceptable carrier.
 7. Thepharmaceutical composition of claim 6 further comprising one or morebacterial capsular polysaccharides or oligosaccharides.
 8. Thepharmaceutical composition of claim 7 wherein the one or more capsularpolysaccharides or oligosaccharides is derived from bacteria selectedfrom the group consisting of N. meningitidis serogroup A, C, Y and/orW-135, Haemophilus influenzae b, Streptococcus pneumoniae, and arepreferably conjugated to a source of T-helper epitopes.